Oligomer-nucleoside phosphate conjugates

ABSTRACT

The invention provides small molecule drugs that are chemically modified by covalent attachment of a water soluble, non-peptidic oligomer. The conjugates of the invention, when administered by any of a number administration routes, exhibits advantages over previously administered compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)to Provisional Application Ser. No. 60/994,768, filed 21 Sep. 2007,which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention provides (among other things) chemically modifiednucleoside phosphates that possess certain advantages over nucleosidephosphates lacking the chemical modification. The chemically modifiednucleoside phosphates described herein relate to and/or haveapplication(s) in (among others) the fields of drug discovery,pharmacotherapy, physiology, organic chemistry and polymer chemistry.

BACKGROUND OF THE INVENTION

When a normal cell loses the ability to control its growth and divisionit is considered cancerous. Cancers or neoplasms have been reported fromevery tissue and cell type. These various cancers are normally treatedusing radiological and/or chemotherapeutic agents. These agents includea wide range of compounds and radioisotopes that work by variousmechanisms. Although development has been directed toward agents capableof selective actions on neoplastic tissues, those available presentlymanifest significant toxicity on normal tissues as a major complicationof therapy.

One class of antineoplastic agents is antimetabolites. A furthersubclass of antimetabolites includes inhibitors of DNA and RNA synthesisand inhibitors of nucleotide synthesis. Many of these are analogs of thenaturally occurring nucleosides: adenosine; guanosine; uridine;cytidine; and thymidine. As these nucleoside analogs are inhibitors ofDNA and/or RNA synthesis or nucleoside metabolism; these compounds arealso useful for treating viral diseases like, including but not limitedto, viral hepatitis, AIDS, common cold, rhinitis, and flu. However,their actions on the normal tissues result in side effects, including,but not limited to, anemia, leucopenia, neutropenia, thrombocytopenia,proteinuria, hematuria, vomiting, pain, fever, rash, dyspnea,constipation, diarrhea, hemorrhage, infection, alopecia, stomatitis,somnolence, paresthesias, chemical arachnoiditis, and neurotoxicity.Adverse events may include death. Thus, there is need in the art toprovide improved nucleoside analogs for treatment of these diseases.

Additionally, Hepatitis C virus infection is one of the leading causesof chronic liver disease; more than 170 million people worldwide areinfected, with HCV genotype 1 predominating in the US. The currentstandard of treatment consists of PEGylated interferon-α2 (pegIFN) aloneor in combination with ribavirin. Combination treatment is effective inonly 50% of patients with HCV genotype 1 infection, and some patientsexperience significant side effects in response to the treatment. Thus,there is considerable interest in the development of more effectiveagents with fewer side effects.

The present invention seeks to address these and other needs in the artby providing (among other things) a conjugate of a water-soluble,non-peptidic oligomer and nucleoside phosphate.

SUMMARY OF THE INVENTION

In one or more embodiments of the invention, compounds are provided; thecompounds comprising a residue of a nucleoside phosphate covalentlyattached either directly or through one or more atoms, to awater-soluble, non-peptidic oligomer.

In one embodiment, a compound is provided comprising a residue of anucleoside phosphate covalently attached, either directly or through oneor more atoms, to a water-soluble, non-peptidic oligomer having anend-capping group selected from hydroxyl and carbon-containingend-capping groups.

In another embodiment, a compound is provided comprising a residue of anucleoside phosphate covalently attached, either directly or through oneor more atoms, to a water-soluble, non-peptidic oligomer having anend-capping group selected from hydroxyl and carbon-containingend-capping groups, having the following structure:

wherein:

-   -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present; and    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group selected from hydroxyl and carbon-containing        end-capping groups.

In another embodiment, a compound is provided comprising a residue of anucleoside phosphate covalently attached, either directly or through oneor more atoms, to a water-soluble, non-peptidic oligomer having anend-capping group selected from hydroxyl and carbon-containingend-capping groups, having the following structure:

wherein:

-   -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present; and    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group selected from hydroxyl and carbon-containing        end-capping groups.

In one or more embodiments, a compound is provided, the compound havingthe following structure:

-   -   base-sugar-phosphate-[X]_(b)-[POLY]_(a)        wherein:    -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present; and    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group selected from hydroxyl and carbon-containing        end-capping groups.

In one or more embodiments, a compound is provided, the compound havingthe following structure:

wherein:

-   -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present; and    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group selected from hydroxyl and carbon-containing        end-capping groups.

In one or more embodiments, a compound is provided, the compound havingthe following structure:

wherein:

-   -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present; and    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group selected from hydroxyl and carbon-containing        end-capping groups.

In one or more embodiments, a compound is provided, the compound havingthe following structure:

-   -   base-sugar-phosphate-[X]_(b)-[POLY]_(a)        wherein:    -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present; and    -   POLY, is a water-soluble, non-peptidic oligomer having an end        capping group selected from hydroxyl and carbon-containing        end-capping groups;    -   with a proviso that the base or the sugar is non-naturally        occurring.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside has a structure encompassed by Formula I.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby Formula II.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby Formula III.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby Formula IV.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby Formula V.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby Formula VI.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby Formula VII.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby Formula VIII.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby Formula IX.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby Formula X.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby Formula XI.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby Formula XII.

In one or more embodiments of the invention, a pharmaceuticalcomposition is provided, the compound comprising a residue of anucleoside phosphate covalently attached via a linker to awater-soluble, non-peptidic oligomer, and, optionally, apharmaceutically acceptable excipient.

In one or more embodiments, a composition is provided, the compositionhaving the following structure:

-   -   base-sugar-phosphate-[X]_(b)-[POLY]_(a)        wherein:    -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present and in each occurrence;    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group;        and, optionally, a pharmaceutically acceptable excipient.

In one or more embodiments of the invention, a dosage form is provided,the dosage form comprising a compound having the following structure:

-   -   base-sugar-phosphate-[X]_(b)-[POLY]_(a)        wherein:    -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present;    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group.

In one or more embodiments of the invention, a method is provided, themethod comprising administering a composition having the followingstructure:

-   -   base-sugar-phosphate-[X]_(b)-[POLY]_(a)        wherein:    -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present;    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group;        and optionally, a pharmaceutically acceptable excipient.

These and other objects, aspects, embodiments and features of theinvention will become more fully apparent when read in conjunction withthe following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

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DETAILED DESCRIPTION OF THE INVENTION

It must be noted that, as used in this specification, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“Water soluble and non-peptidic oligomer” indicates an oligomer that isat least 35% (by weight) soluble, and preferably greater than 95%soluble, in water at room temperature. An unfiltered aqueous preparationof a “water-soluble” oligomer transmits at least 75%, more preferably atleast 95%, of the amount of light transmitted by the same solution afterfiltering. On a weight basis, a “water soluble” oligomer is preferablyat least 35% (by weight) soluble in water, more preferably at least 50%(by weight) soluble in water, still more preferably at least 70% (byweight) soluble in water, and still more preferably at least 85% (byweight) soluble in water. It is preferred, however, that thewater-soluble oligomer is at least 95% (by weight) soluble in water orcompletely soluble in water.

The terms “monomer,” “monomeric subunit” and “monomeric unit” are usedinterchangeably herein and refer to one of the basic structural units ofa polymer or oligomer. In the case of a homo-oligomer, this is definedas a structural repeating unit of the oligomer. In the case of aco-oligomer, a monomeric unit is more usefully defined as the residue ofa monomer which was oligomerized to form the oligomer, since thestructural repeating unit may include more than one type of monomericunit. Preferred oligomers of the invention are homo-oligomers.

An “oligomer” is a molecule possessing from about 1 to about 30monomers. Specific oligomers for use in the invention include thosehaving a variety of geometries such as linear, branched, or forked, tobe described in greater detail below.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Unless otherwise indicated, a“PEG oligomer” or an oligoethylene glycol is one in which substantiallyall (preferably all) monomeric subunits are ethylene oxide subunits,though the oligomer may contain distinct end capping moieties orfunctional groups, e.g., for conjugation. PEG oligomers for use in thepresent invention may comprise of following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. As stated above, for the PEG oligomers, thevariable (n) ranges from 1 to 30, and the terminal groups andarchitecture of the overall PEG can vary. When PEG further comprises afunctional group, A, for linking to, e.g., a small molecule drug, thefunctional group when covalently attached to a PEG oligomer, does notresult in formation of (i) an oxygen-oxygen bond (—O—O—, a peroxidelinkage), or (ii) a nitrogen-oxygen bond (N—O, O—N).

An “end capping group” is generally a non-reactive carbon-containinggroup attached to a terminal oxygen of a PEG oligomer. For the purposesof the present invention, preferred are capping groups having relativelylow molecular weights such as methyl or ethyl. The end-capping group mayalso comprise a detectable label. Such labels include, withoutlimitation, fluorescers, chemiluminescers, moieties used in enzymelabeling, calorimetric labels (e.g., dyes), metal ions, and radioactivemoieties. Another end capping group is hydroxyl.

“Branched”, in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more polymer “arms”extending from a branch point.

“Forked” in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more functional groups(through one or more atoms) extending from a branch point.

A “branch point” refers to a bifurcation point comprising one or moreatoms at which an oligomer branches or forks from a linear structureinto one or more additional arms.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive,” with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions effective to produce a desired reactionin the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group may vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof is meant to encompass protected forms thereof.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively labile bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater may depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include butare not limited to carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides,oligonucleotides, thioesters, thiolesters, and carbonates.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “stable” linkage or bond refers to a chemical bond that issubstantially stable in water, that is to say, does not undergohydrolysis under physiological conditions to any appreciable extent overan extended period of time. Examples of hydrolytically stable linkagesinclude but are not limited to the following: carbon-carbon bonds (e.g.,in aliphatic chains), ethers, amides, urethanes, amines, and the like.Generally, a stable linkage is one that exhibits a rate of hydrolysis ofless than about 1-2% per day under physiological conditions. Hydrolysisrates of representative chemical bonds are found in standard chemistrytextbooks.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater, more preferably 97% or greater, still morepreferably 98% or greater, even more preferably 99% or greater, yetstill more preferably 99.9% or greater, yet still more preferably with99.99% or greater of some given quantity.

“Monodisperse” refers to an oligomer composition wherein substantiallyall of the oligomers in the composition have a well-defined, single(i.e., the same) molecular weight and defined number of monomers, asdetermined by chromatography or mass spectrometry. Monodisperse oligomercompositions are in one sense pure, that is, substantially having asingle and definable number (as a whole number) of monomers rather thana large distribution. A monodisperse oligomer composition possesses aMW/Mn value of 1.0005 or less, and more preferably, a MW/Mn value of1.0000. By extension, a composition comprised of monodisperse conjugatesmeans that substantially all oligomers of all conjugates in thecomposition have a single and definable number (as a whole number) ofmonomers rather than a large distribution and would possess a MW/Mnvalue of 1.0005, and more preferably, a MW/Mn value of 1.0000 if theoligomer were not attached to the moiety derived from a small moleculedrug. A composition comprised of monodisperse conjugates may, however,include one or more nonconjugate substances such as solvents, reagents,excipients, and so forth.

“Bimodal,” in reference to an oligomer composition, refers to anoligomer composition wherein substantially all oligomers in thecomposition have one of two definable and different numbers (as wholenumbers) of monomers rather than a large distribution, and whosedistribution of molecular weights, when plotted as a number fractionversus molecular weight, appears as two separate identifiable peaks.Preferably, for a bimodal oligomer composition as described herein, eachpeak is symmetric about its mean, although the size of the two peaks maydiffer. Ideally, the polydispersity index of each peak in the bimodaldistribution, Mw/Mn, is 1.01 or less, more preferably 1.001 or less, andeven more preferably 1.0005 or less, and even more preferably a MW/Mnvalue of 1.0000. By extension, a composition comprised of bimodalconjugates means that substantially all oligomers of all conjugates inthe composition have one of two definable and different numbers (aswhole numbers) of monomers rather than a large distribution and wouldpossess a MW/Mn value of 1.01 or less, more preferably 1.001 or less andeven more preferably 1.0005 or less, and even more preferably a MW/Mnvalue of 1.0000 if the oligomer were not attached to the moiety derivedfrom a small molecule drug. A composition comprised of bimodalconjugates may, however, include one or more nonconjugate substancessuch as solvents, reagents, excipients, and so forth

A “nucleoside phosphate” refers to an organic, inorganic, ororganometallic compound having a molecular weight of less than about1000 Daltons and having some degree of activity as an antineoplasticagent or as an antiviral agent. In some embodiments of the invention anucleoside or a nitrogenous base is described that may be converted to adesired nucleoside phosphate molecule using the techniques describedherein as well as by the techniques known to one skilled in the art.Nucleoside phosphates of the invention may encompass nucleoside mono-,di-, and tri-phosphates, as well as oligopeptides, oligonucleotides, andother biomolecules having a molecular weight of less than about 1000.

A “biological membrane” is any membrane made of cells or tissues, thatserves as a barrier to at least some foreign entities or otherwiseundesirable materials. As used herein a “biological membrane” includesthose membranes that are associated with physiological protectivebarriers including, for example: the blood-brain barrier; theblood-cerebrospinal fluid barrier; the blood-placental barrier; theblood-milk barrier; the blood-testes barrier; and mucosal barriersincluding the vaginal mucosa, urethral mucosa, anal mucosa, buccalmucosa, sublingual mucosa, and rectal mucosa. Unless the context clearlydictates otherwise, the term “biological membrane” does not includethose membranes associated with the middle gastro-intestinal tract(e.g., stomach and small intestines).

A “biological membrane crossing rate,” provides a measure of acompound's ability to cross a biological barrier, such as theblood-brain barrier (“BBB”). A variety of methods may be used to assesstransport of a molecule across any given biological membrane. Methods toassess the biological membrane crossing rate associated with any givenbiological barrier (e.g., the blood-cerebrospinal fluid barrier, theblood-placental barrier, the blood-milk barrier, the intestinal barrier,and so forth), are known, described herein and/or in the relevantliterature, and/or may be determined by one of ordinary skill in theart.

A “reduced rate of metabolism” refers to a measurable reduction in therate of metabolism of a water-soluble oligomer-small molecule drugconjugate as compared to rate of metabolism of the small molecule drugnot attached to the water-soluble oligomer (i.e., the small moleculedrug itself) or a reference standard material. In the special case of“reduced first pass rate of metabolism,” the same “reduced rate ofmetabolism” is required except that the small molecule drug (orreference standard material) and the corresponding conjugate areadministered orally. Orally administered drugs are absorbed from thegastro-intestinal tract into the portal circulation and may pass throughthe liver prior to reaching the systemic circulation. Because the liveris the primary site of drug metabolism or biotransformation, asubstantial amount of drug may be metabolized before it reaches thesystemic circulation. The degree of first pass metabolism, and thus, anyreduction thereof, may be measured by a number of different approaches.For instance, animal blood samples may be collected at timed intervalsand the plasma or serum analyzed by chromatography/mass spectrometry formetabolite levels. Other techniques for measuring a “reduced rate ofmetabolism” associated with the first pass metabolism and othermetabolic processes are known, described herein and/or in the relevantliterature, and/or may be determined by one of ordinary skill in theart. Preferably, a conjugate of the invention can provide a reduced rateof metabolism reduction satisfying at least one of the following values:at least about 5%, at least about 10%, at least about 15%; least about20%; at least about 25%; at least about 30%; at least about 40%; atleast about 50%; at least about 60%; at least about 70%; at least about80%; and at least about 90%.

A compound (such as a small molecule drug or conjugate thereof) that is“orally bioavailable” is one that exhibits a bioavailability whenadministered orally of greater than 1%, and preferably greater than 10%,where a compound's bioavailability is the fraction of administered drugthat reaches the systemic circulation in unmetabolized form.

“Alkyl” refers to a hydrocarbon chain, ranging from about 1 to 20 atomsin length. Such hydrocarbon chains are preferably but not necessarilysaturated and may be branched or straight chain. Exemplary alkyl groupsinclude methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl,2-ethylpropyl, 3-methylpentyl, and the like. As used herein, “alkyl”includes cycloalkyl when three or more carbon atoms are referenced.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl.

“Non-interfering substituents” are those groups that, when present in amolecule, are non-reactive with other functional groups contained withinthe molecule.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy, etc.),preferably C₁-C₇.

“Links” of an alkyl group are referred to as alkylene, e.g., methylene,ethylene.

The term “substituted” is used throughout the specification. The term“substituted” is defined herein as “encompassing moieties or units thatcan replace a hydrogen atom, two hydrogen atoms, or three hydrogen atomsof a hydrocarbyl moiety. Also, substituted can include replacement ofhydrogen atoms on two adjacent carbons to form a new moiety or unit.”For example, a substituted unit that requires a single hydrogen atomreplacement includes halogen, hydroxyl, and the like. A two hydrogenatom replacement includes carbonyl, oximino, and the like. A twohydrogen atom replacement from adjacent carbon atoms includes epoxy andthe like. Three hydrogen replacement includes cyano and the like. Anepoxide unit is an example of a substituted unit that requiresreplacement of a hydrogen atom on adjacent carbons. The term substitutedis used throughout the present specification to indicate that ahydrocarbyl moiety such as, inter alia, an aromatic ring or alkyl chain,can have one or more of the hydrogen atoms replaced by a substituent.When a moiety is described as “substituted,” any number of the hydrogenatoms may be replaced. For example, 4-hydroxyphenyl is a “substitutedaromatic carbocyclic ring,” (N,N-dimethyl-5-amino)octanyl is a“substituted C₈ alkyl unit,” 3-guanidinopropyl is a “substituted C₃alkyl unit,” and 2-carboxypyridinyl is a “substituted heteroaryl unit.”

The following are non-limiting examples of units which can serve as areplacement for hydrogen atoms when a unit is described as“substituted.”

-   i) —[C(R²⁰)₂]_(p)(CH═CH)_(q)R²⁰; wherein p is from 0 to 12; q is    from 0 to 12;-   ii) —C(Z)R²⁰;-   iii) —C(Z)₂R²⁰;-   iv) —C(Z)CH═CH₂;-   v) —C(Z)N(R²⁰)₂;-   vi) —C(Z)NR²⁰N(R²⁰)₂;-   vii) —CN;-   viii) —CNO;-   ix) —CF₃, —CCl₃, —CBr₃;-   Z) —N(R²⁰)₂;-   xi) —NR²⁰CN;-   xii) —NR²⁰C(Z)R²⁰;-   xiii) —NR²⁰C(Z)N(R²)₂;-   xiv) —NHN(R²)₂;-   xv) —NHOR²⁰;-   xvi) —NCS;-   xvii) —NO₂;-   xviii) —OR²⁰;-   xix) —OCN;-   xx) —OCF₃, —OCCl₃, —OCBr₃;-   xxi) —F, —Br, —I, and mixtures thereof;-   xxii) —SCN;-   xxiii) —SO₃M;-   xxiv) —OSO₃M;-   xxv) —SO₂N(R²⁰)₂;-   xxvi) —SO₂R²⁰;-   xxvii) —P(O)H₂;-   xxviii) —PO₂;-   xxix) —P(O)(OH)₂;-   xxx) and mixtures thereof;-   wherein R²⁰ is hydrogen, substituted or unsubstituted C₁-C₂₀ linear,    branched, or cyclic alkyl, C₆-C₂₀ aryl, and mixtures thereof; M is    hydrogen, or a salt forming cation; Z is ═O, ═S, ═NR²⁰, and mixtures    thereof. Suitable salt forming cations include sodium, lithium,    potassium, calcium, magnesium, ammonium, and the like.

“Electrophile” refers to an ion, atom, or an ionic or neutral collectionof atoms having an electrophilic center, i.e., a center that is electronseeking, capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or an ionic or neutral collectionof atoms having a nucleophilic center, i.e., a center that is seeking anelectrophilic center, and capable of reacting with an electrophile.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that may be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a water-soluble oligomer-small moleculedrug conjugate present in a composition that is needed to provide adesired level of active agent and/or conjugate in the bloodstream or inthe target tissue. The precise amount may depend upon numerous factors,e.g., the particular active agent, the components and physicalcharacteristics of the composition, intended patient population, patientconsiderations, and may readily be determined by one skilled in the art,based upon the information provided herein and available in the relevantliterature.

A “difunctional” oligomer is an oligomer having two functional groupscontained therein, typically at its termini. When the functional groupsare the same, the oligomer is said to be homodifunctional. When thefunctional groups are different, the oligomer is said to beheterodifunctional.

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that may be prevented or treated by administration of aconjugate as described herein and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

As indicated above, the present invention is directed to (among otherthings) a compound comprising a residue of a nucleoside phosphatecovalently attached either directly or through one or more atoms, to awater-soluble, non-peptidic oligomer.

In one embodiment, a compound is provided comprising a residue of anucleoside phosphate covalently attached, either directly or through oneor more atoms, to a water-soluble, non-peptidic oligomer having anend-capping group selected from hydroxyl and carbon-containingend-capping groups.

In one embodiment, a compound is provided comprising a residue of anucleoside phosphate covalently attached, either directly or through oneor more atoms, to a water-soluble, non-peptidic oligomer having anend-capping group other than sulfur-containing end-capping groups.

In another embodiment, a compound is provided comprising a residue of anucleoside phosphate covalently attached, either directly or through oneor more atoms, to a water-soluble, non-peptidic oligomer having anend-capping group selected from hydroxyl and carbon-containingend-capping groups, having the following structure:

wherein:

-   -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present;    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group selected from hydroxyl and carbon-containing        end-capping groups.

In another embodiment, a compound is provided comprising a residue of anucleoside phosphate covalently attached, either directly or through oneor more atoms, to a water-soluble, non-peptidic oligomer having anend-capping group selected from hydroxyl and carbon-containingend-capping groups, having the following structure:

-   -   [POLY]_(a)-[X]_(b)-base-sugar-phosphate        wherein:    -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present;    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group selected from hydroxyl and carbon-containing        end-capping groups.

In one or more embodiments, a compound is provided, the compound havingthe following structure:

-   -   base-sugar-phosphate-[X]_(b)-[POLY]_(a)        wherein:    -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present and in each occurrence;    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group selected from hydroxyl and carbon-containing        end-capping groups.

In one or more embodiments, a compound is provided, the compound havingthe following structure:

wherein:

-   -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present; and    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group selected from hydroxyl and carbon-containing        end-capping groups.

In one or more embodiments, a compound is provided, the compound havingthe following structure:

wherein:

-   -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present; and    -   POLY is a water-soluble, non-peptidic oligomer having an end        capping group selected from hydroxyl and carbon-containing        end-capping groups.

In one or more embodiments, a compound is provided, the compound havingthe following structure:

-   -   base sugar-phosphate-[X]_(b)-[POLY]_(a)        wherein:    -   base is a residue of a small molecule purine or pyrimidine base;    -   (a) is an integer having a value of one or two, inclusive;    -   (b) is an integer having a value of zero or one;    -   X is a linker when present; and    -   POLY, is a water-soluble, non-peptidic oligomer having an end        capping group selected from hydroxyl and carbon-containing        end-capping groups; with a proviso that the base or the sugar is        non-naturally occurring.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is selected from the groupconsisting of N-glycosides of 5-fluorouracil and salts thereof withmedicinally acceptable bases. These and other compounds may besynthesized using the processes known to one skilled in the art and arealso disclosed in U.S. Pat. No. 2,885,396.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is selected from the groupconsisting of 5-fluorouracil nucleotides and salts thereof withpharmaceutically acceptable bases. These and other compounds may besynthesized using the processes known to one skilled in the art and arealso disclosed in U.S. Pat. No. 2,970,139.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is1-(3′,5′-diaroyl-2′-deoxy-D-ribofuranosyl)-5-fluorouracil.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is selected from the groupconsisting of1-(3′,5′-di[p-chlorobenzoyl]-2′-deoxy-D-ribofuranosyl)-5-fluorouracil,1-(3′,5′-di[p-toluoyl]-2′-deoxy-D-ribofuranosyl)-5-fluorouracil, and1-(3′,5′-di[p-toluoyl]-2′-deoxy-D-ribofuranosyl)-thymine. These andother compounds may be synthesized using the processes known to oneskilled in the art and are also disclosed in U.S. Pat. No. 2,949,451.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is 5-fluorouracil and saltsthereof. These and other compounds may be synthesized using theprocesses known to one skilled in the art and are also disclosed in U.S.Pat. No. 2,802,005.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is mono(5-fluorouracilyl)mercury or bis(1-acetyl-5-fluorouracilyl) mercury. These and othercompounds may be synthesized using the processes known to one skilled inthe art and are also disclosed in U.S. Pat. No. 3,041,335.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby the following formula:

wherein R^(x) is a pyridyl group optionally having 1 to 4 substituentsselected from the group consisting of hydroxy group, oxo group, halogenatom, amino group, carboxyl group, cyano group, nitro group, carbamoylgroup, lower alkylcarbamoyl group, carboxy-lower alkylcarbamoyl group,lower alkoxycarbonyl-lower alkylcarbamoyl group, phenyl-carbamoyloptionally substituted with 1 to 3 substituents selected from the groupconsisting of halogen atom, lower alkoxy group and lower alkyl group onthe phenyl ring, lower alkyl group, lower alkenyl group, loweralkoxycarbonyl group, tetrahydrofuranyl group, tetrahydropyranyl group,lower alkoxylower alkyl group, lower alkylthio-lower alkyl group,phenyl-lower alkoxy-lower alkyl group, phthalidyl group and acyloxygroup, and Y is an arylene group selected from the group consisting ofphenylene, naphthalene, pyridinediyl, pyrazinediyl, furandiyl and4-pyridon-1-lower alkyl-diyl; and wherein the acyl moiety of the acyloxygroup is selected from the group consisting of:

(i) C₁-C₂₀ alkenoyl groups optionally substituted with a substituentselected from the group consisting of halogen atom, hydroxy group, loweralkoxy group, aryloxy group, substituted or unsubstituted aryl group,phenyl-lower alkoxycarbonyl group and lower alkylcarbamoyl group,(ii) arylcarbonyl groups optionally substituted with lower alkylenedioxygroup or with 1 to 3 substituents selected from the group consisting ofhalogen atom, lower alkyl group, lower alkoxy group, carboxy group,lower alkoxycarbonyl group, nitro group, cyano group, phenyl-loweralkoxycarbonyl group, hydroxy group, guanidyl group, phenyl-lower alkoxygroup, amino group and amino group substituted with lower alkyl group,(iii) thenylcarbonyl, furanylcarbonyl, thiazolycarbonyl,quinolylcarbonyl, pyrazinylcarbonyl, pyridylcarbonyl,(iv) aryloxycarbonyl groups or straight or branched-chain or cyclicalkoxycarbonyl groups,(v) (C₃-C₈ cycloalkyl) carbonyl groups optionally substituted withhalogen, hydroxy, lower alkoxy or lower alkyl,(vi) lower alkenyl or lower alkynyl carbonyl groups, and(vii) lower alkenyl or lower alkynyl oxycarbonyl groups;and α, is a group which is formed from a 5-fluorouracil derivativelinked by an amide linkage to the carbonyl group to which it isattached, and which is represented by the formula

wherein β represents hydrogen atom, tetrahydrofuranyl, loweralkylcarbamoyl, phthalidyl, lower alkoxy-lower alkyl or loweralkanoyloxy-lower alkoxycarbonyl group.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is3-[3-(6-benzoyloxy-3-cyano-2-pyridyloxycarbonyl)benzoyl]-1-ethoxymethyl-5-fluorouracil.These and other compounds may be synthesized using the processes knownto one skilled in the art and are also disclosed in U.S. Pat. No.4,983,609.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby the following formula:

wherein R is hydrogen, or an acyl radical of the formula R²CO where R²is lower alkyl, phenyl or substituted phenyl groups;R″ is hydrogen;

-   -   R′ where R′ is acyl as defined above or methyl substituted by        one or more phenyl or substituted phenyl groups;        Y is OH, SH, NH₂, NR³R⁴ where R³ and R⁴ are each hydrogen or        lower alkyl or NHOH; and        Z is hydrogen, halogen, lower alkyl, halogenated lower alkyl, or        Nr3 R⁴ where R³ and R⁴ are each hydrogen or lower alkyl.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is2,2′-Anhydro-(1-β-D-arabinofuranosyl)cytosine. These and other compoundsmay be synthesized using the processes known to one skilled in the artand are also disclosed in U.S. Pat. No. 3,463,850.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby the formula:

wherein R represents alkyl.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is5-fluoro-1-hexylcarbamoyluracil. These and other compounds may besynthesized using the processes known to one skilled in the art and arealso disclosed in U.S. Pat. No. 4,071,519.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is azacitidine. These andother compounds may be synthesized using the processes known to oneskilled in the art and are also disclosed in U.S. Pat. No. 3,350,388.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby the following formula:

wherein R is an aliphatic acyl group having 14 to 35 carbon atoms.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside isN-(1-β-D-Arabinofuranosyl-1,2-dihydro-2-oxo-4-pyrimidinyl)docasanamide.These and other compounds may be synthesized using the processes knownto one skilled in the art and are also disclosed in U.S. Pat. No.3,991,045.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is a fully acylated4-thiouracil-1-nucleoside and has a structure encompassed by thefollowing formula:

wherein R₃ is selected from the group consisting of hydrogen, alkylcontaining from 1 to 8 carbon atoms, inclusive, alkenyl containing from3 to 8 carbon atoms, inclusive, cycloalkyl containing from 4 to 8 carbonatoms, inclusive, cycloalkenyl containing from 4 to 8 carbon atoms,inclusive, aryl containing from 6 to 10 carbon atoms, inclusive, aralkylcontaining from 7 to 10 carbon atoms, inclusive, hydroxyl and nitro; andY is a sugar radical containing from 5 to 6 carbon atoms, and whereinthe acyl groups are the acyl radicals of monocarboxylic acids andcontaining from 2 to 12 carbon atoms, inclusive, and halo-, nitro-,hydroxyl-, amino-, cyano-, thiocyano-, and lower-alkoxy-substitutedhydrocarbon monocarboxylic acids containing from 2 to 12 carbon atoms,inclusive.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is4-Amino-1-β-D-arabinofuranosyl-2(1H)-pyrimidinone. These and othercompounds may be synthesized using the processes known to one skilled inthe art and are also disclosed in U.S. Pat. No. 3,116,282.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby the following formula:

wherein R is a base selected from the group consisting of

whereinR¹ is hydrogen, methyl, bromo, fluoro, chloro or iodo;R² is hydroxyl;R³ is hydrogen, bromo, chloro or iodo.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is4-amino-1-[3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1H-pyrimidin-2-one.These and other compounds may be synthesized using the processes knownto one skilled in the art and are also disclosed in U.S. Pat. No.4,808,614.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby the following formula:

wherein R¹, R² and R³ are each independently hydrogen, or an easilyhydrolyzable radical under physiological conditions, with the provisothat, at least one of R¹, R² and R³ is an easily hydrolyzable radicalunder physiological conditions;as well as hydrates or solvates of the compounds of the general formula.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby the following formula:

wherein R¹ is a saturated straight or branched hydrocarbon radicalwherein the number of carbon atoms in the longest straight chain of thishydrocarbon radical ranges from three to seven, or is a radical of theformula —(CH₂)n-^(y) wherein Y is a cyclohexyl radical, a C₁-C₄ alkoxyradical or a phenyl radical and wherein when Y is a cyclohexyl radical nis an integer from 0 to 4, and when Y is C₁-C₄ alkoxy radical or aphenyl radical n is an integer from 2 to 4, and R² is a hydrogen atom ora radical easily hydrolyzable under physiological conditions, or ahydrate or solvate thereof.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is5′-Deoxy-5-fluoro-N-[(pentyloxy)carbonyl]cytidine. These and othercompounds may be synthesized using the processes known to one skilled inthe art and are also disclosed in U.S. Pat. Nos. 4,966,891 and5,472,949.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby the following formula:

wherein X and Y are the same or different and are hydrogen, halogen,OR³, SR³, NR³, R⁴ or NHacyl; R³ and R⁴ being the same or different andbeing hydrogen, a lower alkyl of 1 to 7 carbon atoms, an aralkylcompound selected from the group consisting of benzyl, benzyhydryl ormethoxybenzyl, or an aryl compound selected from the group consisting ofphenyl, chlorophenyl, toluoyl, methoxyphenyl and naphthyl; and NHacylbeing alkanoyl or aroyl amide, alkanoyl being an alkyl carbonyl radicalin which alkyl is a straight or branched chain saturated or unsaturatedhydrocarbon radical having from 1 to 20 carbon atoms; andwherein R¹ and R2 are the same or different and are acyl or aroyl, acylbeing an alkanoyl group of 1 to 20 carbon atoms and aroyl being benzoylor naphthoyl.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby the following formula:

wherein X and Y are the same or different and are hydrogen, halogen,OR³, SR³, NR³R⁴ or NHacyl;R³ and R⁴ being the same or different and being hydrogen, a lower alkylof 1 to 7 carbon atoms, an aralkyl compound selected from the groupconsisting of benzyl, benzhydryl or methoxybenzyl, or an aryl compoundselected from the group consisting of phenyl, chlorophenyl, toluoyl,methoxyphenyl and naphthyl; andNHacyl being alkanoyl or aroyl amide, alkanoyl being an alkyl carbonylradical in which alkyl is a straight or branched chain saturated orunsaturated hydrocarbon radical having from 1 to 20 carbon atoms andaroyl being a benzoyl or naphthoyl; andwherein R¹ and R² are the same or different and are hydrogen, acyl oraroyl, acyl being an alkanoyl group of 1 to 20 carbon atoms and aroylbeing a benzoyl or naphthoyl.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby the following formula:

wherein R, each which may be the same or different, is hydrogen or aprotecting group;wherein Z is a halogen of the group F, Cl, and Br; and pharmaceuticallyacceptable salts thereof, said composition being in combination with apharmaceutically acceptable carrier for oral, topical, or parenteraladministration.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is5-(6-amino-2-chloro-purin-9-yl)-4-fluoro-2-(hydroxymethyl)oxolan-3-ol.These and other compounds may be synthesized using the processes knownto one skilled in the art and are also disclosed in U.S. Pat. Nos.4,751,221, 4,918,179, and 5,384,310.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is9-(5-O-formyl-β-D-arabinofuranosyl)-2-fluoroadenine. These and othercompounds may be synthesized using the processes known to one skilled inthe art and are also disclosed in U.S. Pat. No. 4,357,324.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside has a structure encompassedby the following formula:

wherein X is selected from the class consisting of hydrogen and amino, Ris selected from the class consisting of methyl, benzyl, p-nitrobenzyland hydrogen and R′ is selected from the class consisting of hydrogenand the methyl group.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is6-[(1-methyl-4-nitro-1H-imidazoyl-5-yl)thio]-1H-purin-2-amine. These andother compounds may be synthesized using the processes known to oneskilled in the art and are also disclosed in U.S. Pat. No. 3,056,785.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is2β-D-ribofuranosyl-4-thiazolecarboxamide. These and other compounds maybe synthesized using the processes known to one skilled in the art andare also disclosed in Anticancer Res. 16, 3307-3354 (1996).

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is selected from the groupconsisting of 6-mercaptopurine, 6-purinyl disulfide,2-amino-6-mercaptopurine, 6-thiocyanopurine, 2-amino-6-thiocyanopurine,2-amino-6-iodopurine, 4,5-diamino. These and other compounds may besynthesized using the processes known to one skilled in the art and arealso disclosed in U.S. Pat. Nos. 2,697,709, 2,800,473, 2,884,667,3,019,224, 3,132,144, and 2,721,866.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe nucleoside phosphate or the nucleoside is(R)-3-(2-deoxy-β-D-erythro-pentofuranosyl)-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol.These and other compounds may be synthesized using the processes knownto one skilled in the art and are also disclosed in U.S. Pat. No.3,923,785.

These and other compounds may be synthesized using the processes knownto one skilled in the art and are also disclosed in U.S. Pat. No.3,463,850.

Examples of specific nucleoside phosphate or the nucleosides include,but are not limited to, ancitabine, azacitidine, 6-azauridine,capacitabine, carmofur, cytarabine, decitabine, doxifluridine, emitefur,enocitabine, floxuridine, fluorouracil, gemcitabine, tegafur,cladribine, clofarabine, fludarabine, 6-mercaptopurine, pentostatin,thiamiprine, thioguanine, tiazofurin.

In one or more embodiment of the invention, a compound is provided, thecompound comprising a residue of a nucleoside phosphate covalentlyattached via a linker to a water-soluble, non-peptidic oligomer, whereinthe compound is selected from the group consisting of adenosinephosphate, guanosine phosphate, uridine phosphate, 5-methyluridinephosphate, thymidine phosphate, cytidine phosphate, deoxyadenosinephosphate, deoxyguanosine phosphate, deoxyuridine phosphate,deoxy-5-methyluridine phosphate, deoxythymidine phosphate, deoxycytidinephosphate, xanthosine phosphate, deoxyxanthosine phosphate,pseudouridine phosphate, deoxypseudouridine phosphate, orotidinephosphate, deoxyorotidine phosphate, inosine phosphate, deoxyinosinephosphate, nicatinamide adenine dinucleotide, flavin adeninedinucleotide, falvin mononucleotide, nicotinamide mononucleotide,nicotinamide adenine dinucleotide phosphate.

It is believed that an advantage of the conjugates of the presentinvention is their ability to retain some degree of nucleoside phosphateactivity while also exhibiting a decrease in metabolism. Although notwishing to be bound by theory, it is believed that theoligomer-containing conjugates described herein, in contrast to theunconjugated “original” nucleoside phosphate, are not metabolized asreadily because the oligomer serves to reduce the overall affinity ofthe compound to substrates that may metabolize nucleoside phosphates. Inaddition (and again, not wishing to be bound by theory), the extra sizeintroduced by the oligomer, in contrast to the unconjugated “original”nucleoside phosphate, reduces the ability of the compound to cross theblood-brain barrier. Even should the linkage between the residue of thenucleoside phosphate and the oligomer be degradable, the compound stilloffers advantages (such as avoiding first-pass metabolism upon initialabsorption).

Use of discrete oligomers (e.g., from a monodisperse or bimodalcomposition of oligomers, in contrast to relatively impure compositions)to form conjugates may advantageously alter certain propertiesassociated with the corresponding small molecule drug. For instance, aconjugate of the invention, when administered by any of a number ofsuitable administration routes, such as parenteral, oral, transdermal,buccal, pulmonary, or nasal, exhibits reduced penetration across theblood-brain barrier. It is preferred that the conjugates exhibit slowed,minimal or effectively no crossing of the blood-brain barrier, whilestill crossing the gastro-intestinal (GI) walls and into the systemiccirculation if oral delivery is intended. Moreover, the conjugates ofthe invention maintain a degree of bioactivity as well asbioavailability in their conjugated form.

With respect to the blood-brain barrier (“BBB”), this barrier restrictsthe transport of drugs from the blood to the brain. This barrierconsists of a continuous layer of unique endothelial cells joined bytight junctions. The cerebral capillaries, which comprise more than 95%of the total surface area of the BBB, represent the principal route forthe entry of solutes and drugs into the central nervous system.

For compounds whose degree of blood-brain barrier crossing ability isnot known, the ability may be determined using a suitable animal modelsuch as an in situ rat brain perfusion (“RBP”) model as describedherein. Briefly, the RBP technique involves cannulation of the carotidartery followed by perfusion with a compound solution under controlledconditions, followed by a wash out phase to remove compound remaining inthe vascular space. (Such analyses may be conducted, for example, bycontract research organizations such as Absorption Systems, Exton, Pa.).More specifically, in the RBP model, a cannula is placed in the leftcarotid artery and the side branches are tied off. A physiologic buffercontaining the compound (5 micromolar) is perfused at a flow rate of 10mL/min in a single pass perfusion experiment. After 30 seconds, theperfusion is stopped and the brain vascular contents are washed out withcompound-free buffer for an additional 30 seconds. The brain tissue isthen removed and analyzed for compound concentrations via liquidchromatograph with tandem mass spectrometry detection (LC/MS/MS).Alternatively, blood-brain barrier permeability can be estimated basedupon a calculation of the compound's molecular polar surface area(“PSA”), which is defined as the sum of surface contributions of polaratoms (usually oxygens, nitrogens and attached hydrogens) in a molecule.The PSA has been shown to correlate with compound transport propertiessuch as blood-brain barrier transport. Methods for determining acompound's PSA can be found, e.g., in, Ertl, P., et al., J. Med. Chem.2000, 43, 3714-3717; and Kelder, J., et al., Pharm. Res. 1999, 16,1514-1519.

With respect to the blood-brain barrier, the water-solubleoligomer-small molecule drug conjugate exhibits a blood-brain barriercrossing rate that is reduced as compared to the crossing rate of thesmall molecule drug not attached to the water-soluble oligomer.Exemplary reductions in blood-brain barrier crossing rates for theconjugates described herein include reductions of: at least about 5%; atleast about 10%; at least about 25%; at least about 30%; at least about40%; at least about 50%; at least about 60%; at least about 70%; atleast about 80%; or at least about 90%, when compared to the blood-brainbarrier crossing rate of the small molecule drug not attached to thewater-soluble oligomer. A preferred reduction in the blood-brain barriercrossing rate for a conjugate is at least about 20%.

As indicated above, the compounds of the invention include a residue ofa nucleoside phosphate. Assays for determining whether a given compoundmay inhibit growth are described infra.

The nucleoside phosphates used in the conjugates are small moleculedrugs, that is to say, pharmacologically active compounds having amolecular weight of less than about 1000 Daltons. Small molecule drugs,for the purpose of the invention, include oligopeptides,oligonucleotides, and other biomolecules having a molecular weight ofless than about 1000 Daltons. Also encompassed in the term “smallmolecule drug” is any fragment of a peptide, protein or antibody,including native sequences and variants falling within the molecularweight range stated above. In one or more embodiments, however, it ispreferred that the small molecule drug satisfies one or more of thefollowing: not an oligopeptide; not an oligonucleotide; not an antibody;and not a fragment of any of the foregoing.

Exemplary molecular weights of small molecule drugs include molecularweights of: less than about 950; less than about 900; less than about850; less than about 800; less than about 750; less than about 700; lessthan about 650; less than about 600; less than about 550; less thanabout 500; less than about 450; less than about 400; less than about350; and less than about 300.

The small molecule drug used in the invention, if chiral, may beobtained from a racemic mixture, or an optically active form, forexample, a single optically active enantiomer, or any combination orratio of enantiomers. In addition, the small molecule drug may possessone or more geometric isomers. With respect to geometric isomers, acomposition may a mixture of two or more geometric isomers. A smallmolecule drug for use in the present invention may be in its customaryactive mode, or may possess some degree of modification. For example, asmall molecule drug may have a targeting agent, tag, or transporterattached thereto, prior to or after covalent attachment of an oligomer.Alternatively, the small molecule drug may possess a lipophilic moietyattached thereto, such as a phospholipid (e.g.,distearoylphosphatidylethanolamine or “DSPE,”dipalmitoylphosphatidylethanolamine or “DPPE,” and so forth) or a smallfatty acid. In some instances, however, it is preferred that the smallmolecule drug moiety does not include attachment to a lipophilic moiety.

The nucleoside phosphate for coupling to a water-soluble, non-peptidicoligomer possesses a free hydroxyl, carboxyl, thio, amino group, or thelike (i.e., “handle”) suitable for covalent attachment to the oligomer.In addition, the nucleoside phosphate may be modified by introduction ofa reactive group, preferably by conversion of one of its existingfunctional groups to a functional group suitable for formation of astable covalent linkage between the oligomer and the drug. Bothapproaches are illustrated in the Experimental section.

The water-soluble, non-peptidic oligomer comprises one or more monomersserially attached to form a chain of monomers. The oligomer may beformed from a single monomer type (i.e., is homo-oligomeric) or two orthree monomer types (i.e., is co-oligomeric). Preferably, each oligomeris a co-oligomer of two monomers or, more preferably, is ahomo-oligomer.

Accordingly, each oligomer is composed of up to three different monomertypes selected from the group consisting of: alkylene oxide, such asethylene oxide or propylene oxide; olefinic alcohol, such as vinylalcohol, 1-propenol or 2-propenol; vinyl pyrrolidone; hydroxyalkylmethacrylamide or hydroxyalkyl methacrylate, where alkyl is preferablymethyl; α-hydroxy acid, such as lactic acid or glycolic acid;phosphazene, oxazoline, amino acids, carbohydrates such asmonosaccharides, saccharide or mannitol; and N-acryloylmorpholine.Preferred monomer types include alkylene oxide, olefinic alcohol,hydroxyalkyl methacrylamide or methacrylate, N-acryloylmorpholine, andα-hydroxy acid. Preferably, each oligomer is, independently, aco-oligomer of two monomer types selected from this group, or, morepreferably, is a homo-oligomer of one monomer type selected from thisgroup.

The two monomer types in a co-oligomer may be of the same monomer type,for example, two alkylene oxides, such as ethylene oxide and propyleneoxide. Preferably, the oligomer is a homo-oligomer of ethylene oxide.Usually, although not necessarily, the terminus (or termini) of theoligomer that is not covalently attached to a small molecule is cappedto render it unreactive. Alternatively, the terminus may include areactive group. When the terminus is a reactive group, the reactivegroup is either selected such that it is unreactive under the conditionsof formation of the final oligomer or during covalent attachment of theoligomer to a small molecule drug, or it is protected as necessary. Onecommon end-functional group is hydroxyl or —OH, particularly foroligoethylene oxides.

The water-soluble, non-peptidic oligomer (e.g., “POLY” in variousstructures provided herein) may have any of a number of differentgeometries. For example, the water-soluble, non-peptidic oligomer may belinear, branched, or forked. The water-soluble, non-peptidic oligomer islinear or is branched, for example, having one branch point. Althoughmuch of the discussion herein is focused upon poly(ethylene oxide) as anillustrative oligomer, the discussion and structures presented hereincan be readily extended to encompass any of the water-soluble,non-peptidic oligomers described above.

The molecular weight of the water-soluble, non-peptidic oligomer,excluding the linker portion, is generally relatively low. Exemplaryvalues of the molecular weight of the water-soluble polymer include:below about 1500; below about 1450; below about 1400; below about 1350;below about 1300; below about 1250; below about 1200; below about 1150;below about 1100; below about 1050; below about 1000; below about 950;below about 900; below about 850; below about 800; below about 750;below about 700; below about 650; below about 600; below about 550;below about 500; below about 450; below about 400; below about 350;below about 300; below about 250; below about 200; and below about 100Daltons.

Exemplary ranges of molecular weights of the water-soluble, non-peptidicoligomer (excluding the linker) include: from about 100 to about 1400Daltons; from about 100 to about 1200 Daltons; from about 100 to about800 Daltons; from about 100 to about 500 Daltons; from about 100 toabout 400 Daltons; from about 200 to about 500 Daltons; from about 200to about 400 Daltons; from about 75 to 1000 Daltons; and from about 75to about 750 Daltons.

Preferably, the number of monomers in the water-soluble, non-peptidicoligomer falls within one or more of the following ranges: between about1 and about 30 (inclusive); between about 1 and about 25; between about1 and about 20; between about 1 and about 15; between about 1 and about12; between about 1 and about 10. In certain instances, the number ofmonomers in series in the oligomer (and the corresponding conjugate) isone of 1, 2, 3, 4, 5, 6, 7, or 8. In additional embodiments, theoligomer (and the corresponding conjugate) contains 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 monomers in series. In yet furtherembodiments, the oligomer (and the corresponding conjugate) possesses21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 monomers in series. Thus, forexample, when the water-soluble, non-peptidic polymer includesCH₃—(OCH₂CH₂)_(n)—, “n” is an integer that may be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30, and may fall within one or more of the followingranges: between about 1 and about 25; between about 1 and about 20;between about 1 and about 15; between about 1 and about 12; betweenabout 1 and about 10.

When the water-soluble, non-peptidic oligomer has 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 monomers, these values correspond to a methoxy end-cappedoligo(ethylene oxide) having a molecular weights of about 75, 119, 163,207, 251, 295, 339, 383, 427, and 471 Daltons, respectively. When theoligomer has 11, 12, 13, 14, or 15 monomers, these values correspond tomethoxy end-capped oligo(ethylene oxide) having molecular weightscorresponding to about 515, 559, 603, 647, and 691 Daltons,respectively.

When the water-soluble, non-peptidic oligomer is attached to thenucleoside phosphate (in contrast to the step-wise addition of one ormore monomers to effectively “grow” the oligomer onto the nucleosidephosphate), it is preferred that the composition containing an activatedform of the water-soluble, non-peptidic oligomer be monodispersed. Inthose instances, however, where a bimodal composition is employed, thecomposition will possess a bimodal distribution centering around any twoof the above numbers of monomers. Ideally, the polydispersity index ofeach peak in the bimodal distribution, Mw/Mn, is 1.01 or less, and evenmore preferably, is 1.001 or less, and even more preferably is 1.0005 orless. More preferably, each peak possesses a MW/Mn value of 1.0000. Forinstance, a bimodal oligomer may have any one of the following exemplarycombinations of monomer subunits: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, and so forth; 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, and soforth; 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, and so forth; 4-5, 4-6, 4-7,4-8, 4-9, 4-10, and so forth; 5-6, 5-7, 5-8, 5-9, 5-10, and so forth;6-7, 6-8, 6-9, 6-10, and so forth; 7-8, 7-9, 7-10, and so forth; and8-9, 8-10, and so forth.

In some instances, the composition containing an activated form of thewater-soluble, non-peptidic oligomer will be trimodal or eventetramodal, possessing a range of monomers units as previouslydescribed. Oligomer compositions possessing a well-defined mixture ofoligomers (i.e., being bimodal, trimodal, tetramodal, and so forth) canbe prepared by mixing purified monodisperse oligomers to obtain adesired profile of oligomers (a mixture of two oligomers differing onlyin the number of monomers is bimodal; a mixture of three oligomersdiffering only in the number of monomers is trimodal; a mixture of fouroligomers differing only in the number of monomers is tetramodal), oralternatively, can be obtained from column chromatography of apolydisperse oligomer by recovering the “center cut”, to obtain amixture of oligomers in a desired and defined molecular weight range.

It is preferred that the water-soluble, non-peptidic oligomer isobtained from a composition that is preferably unimolecular ormonodisperse. That is, the oligomers in the composition possess the samediscrete molecular weight value rather than a distribution of molecularweights. Some monodisperse oligomers may be purchased or prepared fromcommercial sources (e.g., Sigma-Aldrich, St. Louis, Mo.), oralternatively, may be chemically synthesized. Water-soluble,non-peptidic oligomers may be prepared as described in Chen Y., Baker,G. L., J. Org. Chem., 6870-6873 (1999), WO 02/098949, and U.S. PatentApplication Publication 2005/0136031.

When present, the linker or linkage (through which the water-soluble,non-peptidic polymer is attached to the nucleoside phosphate) may be asingle atom, such as an oxygen or a sulfur, two atoms, or a number ofatoms. A linker may be linear in nature. The linkage, “X” ishydrolytically stable, and is preferably also enzymatically stable.Preferably, the linkage “X” is one having a chain length of less thanabout 12 atoms, and preferably less than about 10 atoms, and even morepreferably less than about 8 atoms and even more preferably less thanabout 5 atoms, whereby length is meant the number of atoms in a singlechain, not counting substituents. For instance, a urea linkage such asthis, R_(ohgomer)-NH—(C═O)—NH—R′_(drug), is considered to have a chainlength of 3 atoms (—NH—C(O)—NH—). In selected embodiments, the linkagedoes not comprise further spacer groups.

In some instances, the linker “X” comprises an ether, amide, urethane,amine, thioether, urea, or a carbon-carbon bond. Functional groups suchas those discussed below, are used for forming the linkages. The linkagemay less preferably also comprise (or be adjacent to or flanked by)spacer groups, as described further below. Spacers are useful ininstances where the bioactivity of the conjugate is significantlyreduced due to the positioning of the oligomer on the parent drug.

More specifically, in selected embodiments, a linker of the invention,X, may be any of the following: “—” (i.e., a covalent bond, that may bestable or degradable, between the residue of the small moleculenucleoside phosphate and the water-soluble, non-peptidic oligomer), —O—,—NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —CH₂—C(O)O—, —CH₂—OC(O)—,—C(O)O—CH₂—, —OC(O)—CH₂—, C(O)—NH, NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH₂—,—CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—,—O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂, —CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group,—N(R)—, R is selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl and substituted aryl.

For purposes of the present invention, however, a series of atoms is notconsidered as a linkage when the series of atoms is immediately adjacentto an oligomer segment, and the series of atoms is but another monomersuch that the proposed linkage would represent a mere extension of theoligomer chain.

The linkage “X” between the water-soluble, non-peptidic oligomer and thesmall molecule is formed by reaction of a functional group on a terminusof the oligomer (or one or more monomers when it is desired to “grow”the oligomer onto the nucleoside phosphate) with a correspondingfunctional group within the nucleoside phosphate. Illustrative reactionsare described briefly below. For example, an amino group on an oligomermay be reacted with a carboxylic acid or an activated carboxylic acidderivative on the small molecule, or vice versa, to produce an amidelinkage. Alternatively, reaction of an amine on an oligomer with anactivated carbonate (e.g. succinimidyl or benzotriazyl carbonate) on thedrug, or vice versa, forms a carbamate linkage. Reaction of an amine onan oligomer with an isocyanate (R—N═C═O) on a drug, or vice versa, formsa urea linkage (R—NH—(C═O)—NH—R′). Further, reaction of an alcohol(alkoxide) group on an oligomer with an alkyl halide, or halide groupwithin a drug, or vice versa, forms an ether linkage. In yet anothercoupling approach, a small molecule having an aldehyde function iscoupled to an oligomer amino group by reductive amination, resulting information of a secondary amine linkage between the oligomer and thesmall molecule.

A particularly preferred water-soluble, non-peptidic oligomer is anoligomer bearing an aldehyde functional group. In this regard, theoligomer may have the following structure:CH₃O—(CH₂—CH₂—O)_(n)—(CH₂)_(p)—C(O)H, wherein (n) is one of 1, 2, 3, 4,5, 6, 7, 8, 9 and 10 and (p) is one of 1, 2, 3, 4, 5, 6 and 7. Preferred(n) values include 3, 5 and 7 and preferred (p) values 2, 3 and 4.

The terminus of the water-soluble, non-peptidic oligomer not bearing afunctional group may be capped to render it unreactive. When theoligomer does includes a further functional group at a terminus otherthan that intended for formation of a conjugate, that group is eitherselected such that it is unreactive under the conditions of formation ofthe linkage “X,” or it is protected during the formation of the linkage“X.”

As stated above, the water-soluble, non-peptidic oligomer includes atleast one functional group prior to conjugation. The functional groupcomprises an electrophilic or nucleophilic group for covalent attachmentto a small molecule, depending upon the reactive group contained withinor introduced into the small molecule. Examples of nucleophilic groupsthat may be present in either the oligomer or the small molecule includehydroxyl, amine, hydrazine (—NHNH₂), hydrazide (—C(O)NHNH₂), and thiol.Preferred nucleophiles include amine, hydrazine, hydrazide, and thiol,particularly amine. Small molecule drugs for covalent attachment to anoligomer may possess a free hydroxyl, amino, thio, aldehyde, ketone, orcarboxyl group.

Examples of electrophilic functional groups that may be present ineither the oligomer or the small molecule include carboxylic acid,carboxylic ester, particularly imide esters, orthoester, carbonate,isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate,methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy,sulfonate, thiosulfonate, silane, alkoxysilane, and halosilane. Morespecific examples of these groups include succinimidyl ester orcarbonate, imidazoyl ester or carbonate, benzotriazole ester orcarbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyldisulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, andtresylate (2,2,2-trifluoroethanesulfonate).

Also included are sulfur analogs of several of these groups, such asthione, thione hydrate, thioketal, is 2-thiazolidine thione, etc., aswell as hydrates or protected derivatives of any of the above moieties(e.g. aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal,ketal, thioketal, thioacetal).

An “activated derivative” of a carboxylic acid refers to a carboxylicacid derivative which reacts readily with nucleophiles, generally muchmore readily than the underivatized carboxylic acid. Activatedcarboxylic acids include, for example, acid halides (such as acidchlorides), anhydrides, carbonates, and esters. Such esters includeimide esters, of the general form —(CO)O—N[(CO)—]₂; for example,N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters. Alsopreferred are imidazolyl esters and benzotriazole esters. Particularlypreferred are activated propionic acid or butanoic acid esters, asdescribed in co-owned U.S. Pat. No. 5,672,662. These include groups ofthe form —(CH₂)₂₋₃C(═O)O-Q, where Q is preferably selected fromN-succinimide, N-sulfosuccinimide, N-phthalimide, N-glutarimide,N-tetrahydrophthalimide, N-norbornene-2,3-dicarboximide, benzotriazole,7-azabenzotriazole, and imidazole.

Other preferred electrophilic groups include succinimidyl carbonate,maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate,p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyldisulfide.

These electrophilic groups are subject to reaction with nucleophiles,e.g. hydroxy, thio, or amino groups, to produce various bond types.Preferred for the present invention are reactions which favor formationof a hydrolytically stable linkage. For example, carboxylic acids andactivated derivatives thereof, which include orthoesters, succinimidylesters, imidazolyl esters, and benzotriazole esters, react with theabove types of nucleophiles to form esters, thioesters, and amides,respectively, of which amides are the more hydrolytically stable. Asmentioned above, more preferred are conjugates having a hydrolyticallystable linkage between the oligomer and the drug. Carbonates, includingsuccinimidyl, imidazolyl, and benzotriazole carbonates, react with aminogroups to form carbamates. Isocyanates (R—N═C═O) react with hydroxyl oramino groups to form, respectively, carbamate (RNH—C(O)—OR′) or urea(RNH—C(O)—NHR′) linkages. Aldehydes, ketones, glyoxals, diones and theirhydrates or alcohol adducts (i.e. aldehyde hydrate, hemiacetal, acetal,ketone hydrate, hemiketal, and ketal) are preferably reacted withamines, followed by reduction of the resulting imine, if desired, toprovide an amine linkage (reductive amination).

Several of the electrophilic functional groups include electrophilicdouble bonds to which nucleophilic groups, such as thiols, may be added,to form, for example, thioether bonds. These groups include maleimides,vinyl sulfones, vinyl pyridine, acrylates, methacrylates, andacrylamides. Other groups comprise leaving groups which may be displacedby a nucleophile; these include chloroethyl sulfone, pyridyl disulfides(which include a cleavable S—S bond), iodoacetamide, mesylate, tosylate,thiosulfonate, and tresylate. Epoxides react by ring opening by anucleophile, to form, for example, an ether or amine bond. Reactionsinvolving complementary reactive groups such as those noted above on theoligomer and the small molecule are utilized to prepare the conjugatesof the invention.

In some instances the nucleoside phosphate may not have a functionalgroup suited for conjugation. In this instance, it is possible to modifythe “original” nucleoside phosphate so that it does have a functionalgroup suited for conjugation. For example, if the nucleoside phosphatehas an amide group, but an amine group is desired, it is possible tomodify the amide group to an amine group by way of a Hofmannrearrangement, Curtius rearrangement (once the amide is converted to anazide) or Lossen rearrangement (once amide is concerted to hydroxamidefollowed by treatment with tolyene-2-sulfonyl chloride/base).

It is possible to prepare a conjugate of small molecule nucleosidephosphate bearing a carboxyl group wherein the carboxyl group-bearingsmall molecule nucleoside phosphate is coupled to an amino-terminatedoligomeric ethylene glycol, to provide a conjugate having an amide groupcovalently linking the small molecule nucleoside phosphate to theoligomer. This can be performed, for example, by combining the carboxylgroup-bearing small molecule nucleoside phosphate with theamino-terminated oligomeric ethylene glycol in the presence of acoupling reagent, (such as dicyclohexylcarbodiimide or “DCC”) in ananhydrous organic solvent.

Further, it is possible to prepare a conjugate of a small moleculenucleoside phosphate bearing a hydroxyl group wherein the hydroxylgroup-bearing small molecule nucleoside phosphate is coupled to anoligomeric ethylene glycol halide to result in an ether (—O—) linkedsmall molecule conjugate. This can be performed, for example, by usingsodium hydride to deprotonate the hydroxyl group followed by reactionwith a halide-terminated oligomeric ethylene glycol.

In another example, it is possible to prepare a conjugate of a smallmolecule nucleoside phosphate bearing a ketone group by first reducingthe ketone group to form the corresponding hydroxyl group. Thereafter,the small molecule nucleoside phosphate now bearing a hydroxyl group canbe coupled as described herein.

In still another instance, it is possible to prepare a conjugate of asmall molecule nucleoside phosphate bearing an amine group. In oneapproach, the amine group-bearing small molecule nucleoside phosphateand an aldehyde-bearing oligomer are dissolved in a suitable bufferafter which a suitable reducing agent (e.g., NaCNBH₃) is added.Following reduction, the result is an amine linkage formed between theamine group of the amine group-containing small molecule nucleosidephosphate and the carbonyl carbon of the aldehyde-bearing oligomer.

In another approach for preparing a conjugate of a small moleculenucleoside phosphate bearing an amine group, a carboxylic acid-bearingoligomer and the amine group-bearing small molecule nucleoside phosphateare combined, in the presence of a coupling reagent (e.g., DCC). Theresult is an amide linkage formed between the amine group of the aminegroup-containing small molecule nucleoside phosphate and the carbonyl ofthe carboxylic acid-bearing oligomer.

The conjugates of the invention can exhibit a reduced blood-brainbarrier crossing rate. Moreover, the conjugates maintain at least about5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more of the bioactivity of theunmodified parent small molecule drug.

The selection of an optimally sized oligomer can be determined asfollows.

First, an oligomer obtained from a monodisperse or bimodal water solubleoligomer is conjugated to the small molecule drug. Preferably, the drugis orally bioavailable, and on its own, exhibits a blood-brain barriercrossing rate. Next, the ability of the conjugate to cross theblood-brain barrier is determined using an appropriate model andcompared to that of the unmodified parent drug. If the results arefavorable, that is to say, if, for example, the rate of crossing issignificantly reduced, then the bioactivity of conjugate is furtherevaluated. In one or more embodiments, the drug in conjugated form canbe bioactive, and preferably, maintains a significant degree ofbioactivity relative to the parent drug, i.e., greater than about 30% ofthe bioactivity of the parent drug, or even more preferably, greaterthan about 50% of the bioactivity of the parent drug.

Then, the above steps are repeated using oligomers of the same monomertype but having a different number of subunits.

For each conjugate whose ability to cross the blood-brain barrier isreduced in comparison to the non-conjugated small molecule drug, itsoral bioavailability is then assessed. Based upon these results ofsequential addition of increasing numbers of discrete monomers to agiven small molecule at a given position or location within the smallmolecule, it is possible to determine the size of the oligomer effectivein providing a conjugate having an optimal balance between reduction inbiological membrane crossing, oral bioavailability, and bioactivity. Thesmall size of the oligomers may make such screenings feasible, and mayallow one to effectively tailor the properties of the resultingconjugate. By making small, incremental changes in oligomer size, andutilizing an experimental design approach, one can effectively identifya conjugate having a favorable balance of reduction in biologicalmembrane crossing rate, bioactivity, and oral bioavailability. In someinstances, attachment of an oligomer as described herein is effective toactually increase oral bioavailability of the drug.

For example, one of ordinary skill in the art, using routineexperimentation, may determine a best suited molecular size and linkagefor improving oral bioavailability by first preparing a series ofoligomers with different weights and functional groups and thenobtaining the necessary clearance profiles by administering theconjugates to a patient and taking periodic blood and/or urine sampling.Once a series of clearance profiles have been obtained for each testedconjugate, a suitable conjugate can be identified.

Animal models (rodents and dogs) may also be used to study drugtransport and bioavailability. In addition, non-in vivo methods includerodent everted gut excised tissue and Caco-2 cell monolayertissue-culture models. These models are useful in predicting oral drugbioavailability.

The conjugates of the invention can then be evaluated for theirbiological activities, including but not limited to, antiviral activity,anti-neoplastic activity, anti-angiogenic activity and such. Such assaysare known to one skilled in the art and are also disclosed in theexperimental section herein. Further, such assays are also disclosed inthe US and other foreign patents that are referenced herein and areherein incorporated by reference in their entirety.

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself may be in asolid form (e.g., a precipitate), which may be combined with a suitablepharmaceutical excipient that may be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient may also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant may be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that may be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The amount of the conjugate in the composition may vary depending on anumber of factors, but may optimally be a therapeutically effective dosewhen the composition is stored in a unit dose container. Atherapeutically effective dose may be determined experimentally byrepeated administration of increasing amounts of the conjugate in orderto determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition may varydepending on the activity of the excipient and particular needs of thecomposition. The optimal amount of any individual excipient isdetermined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

The excipient may be present in the composition in an amount of about 1%to about 99% by weight, preferably from about 5%-98% by weight, morepreferably from about 15-95% by weight of the excipient, withconcentrations less than 30% by weight more preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., Handbook of Pharmaceutical Excipients, Edition, AmericanPharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions may take any number of forms and theinvention is not limited in this regard. Exemplary preparations are morepreferably in a form suitable for oral administration such as a tablet,caplet, capsule, gel cap, troche, dispersion, suspension, solution,elixir, syrup, lozenge, transdermal patch, spray, suppository, andpowder.

Oral dosage forms are preferred for those conjugates that are orallyactive, and include tablets, caplets, capsules, gel caps, suspensions,solutions, elixirs, and syrups, and may also comprise a plurality ofgranules, beads, powders or pellets that are optionally encapsulated.Such dosage forms are prepared using conventional methods known to thosein the field of pharmaceutical formulation and described in thepertinent texts.

Tablets and caplets, for example, may be manufactured using standardtablet processing procedures and equipment. Direct compression andgranulation techniques are preferred when preparing tablets or capletscontaining the conjugates described herein. In addition to theconjugate, the tablets and caplets may generally contain inactive,pharmaceutically acceptable carrier materials such as binders,lubricants, disintegrants, fillers, stabilizers, surfactants, coloringagents, and the like. Binders are used to impart cohesive qualities to atablet, and thus ensure that the tablet remains intact. Suitable bindermaterials include, but are not limited to, starch (including corn starchand pregelatinized starch), gelatin, sugars (including sucrose, glucose,dextrose and lactose), polyethylene glycol, waxes, and natural andsynthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone,cellulosic polymers (including hydroxypropyl cellulose, hydroxypropylmethylcellulose, methyl cellulose, microcrystalline cellulose, ethylcellulose, hydroxyethyl cellulose, and the like), and Veegum. Lubricantsare used to facilitate tablet manufacture, promoting powder flow andpreventing particle capping (i.e., particle breakage) when pressure isrelieved. Useful lubricants are magnesium stearate, calcium stearate,and stearic acid. Disintegrants are used to facilitate disintegration ofthe tablet, and are generally starches, clays, celluloses, algins, gums,or crosslinked polymers. Fillers include, for example, materials such assilicon dioxide, titanium dioxide, alumina, talc, kaolin, powderedcellulose, and microcrystalline cellulose, as well as soluble materialssuch as mannitol, urea, sucrose, lactose, dextrose, sodium chloride, andsorbitol. Stabilizers, as well known in the art, are used to inhibit orretard drug decomposition reactions that include, by way of example,oxidative reactions.

Capsules are also preferred oral dosage forms, in which case theconjugate-containing composition may be encapsulated in the form of aliquid or gel (e.g., in the case of a gel cap) or solid (includingparticulates such as granules, beads, powders or pellets). Suitablecapsules include hard and soft capsules, and are generally made ofgelatin, starch, or a cellulosic material. Two-piece hard gelatincapsules are preferably sealed, such as with gelatin bands or the like.

Included are parenteral formulations in the substantially dry form (as alyophilizate or precipitate, which may be in the form of a powder orcake), as well as formulations prepared for injection, which are liquidand requires the step of reconstituting the dry form of parenteralformulation. Examples of suitable diluents for reconstituting solidcompositions prior to injection include bacteriostatic water forinjection, dextrose 5% in water, phosphate-buffered saline, Ringer'ssolution, saline, sterile water, deionized water, and combinationsthereof.

In some cases, compositions intended for parenteral administration maytake the form of nonaqueous solutions, suspensions, or emulsions, eachbeing sterile. Examples of nonaqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate.

The parenteral formulations described herein may also contain adjuvantssuch as preserving, wetting, emulsifying, and dispersing agents. Theformulations are rendered sterile by incorporation of a sterilizingagent, filtration through a bacteria-retaining filter, irradiation, orheat.

The conjugate may also be administered through the skin usingconventional transdermal patch or other transdermal delivery system,wherein the conjugate is contained within a laminated structure thatserves as a drug delivery device to be affixed to the skin. In such astructure, the conjugate is contained in a layer, or “reservoir,”underlying an upper backing layer. The laminated structure may contain asingle or multiple reservoirs.

The conjugate may also be formulated into a suppository for rectaladministration. With respect to suppositories, the conjugate is mixedwith a suppository base material which is (e.g., an excipient thatremains solid at room temperature but softens, melts or dissolves atbody temperature) such as coca butter (theobroma oil), polyethyleneglycols, glycerinated gelatin, fatty acids, and combinations thereof.Suppositories may be prepared by, for example, performing the followingsteps (not necessarily in the order presented): melting the suppositorybase material to form a melt; incorporating the conjugate (either beforeor after melting of the suppository base material); pouring the meltinto a mold; cooling the melt (e.g., placing the melt-containing mold ina room temperature environment) to thereby form suppositories; andremoving the suppositories from the mold.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with the conjugate. The method comprisesadministering, generally orally, a therapeutically effective amount ofthe conjugate (preferably provided as part of a pharmaceuticalpreparation). Other modes of administration are also contemplated, suchas pulmonary, nasal, buccal, rectal, sublingual, transdermal, andparenteral. As used herein, the term “parenteral” includes subcutaneous,intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal,and intramuscular injection, as well as infusion injections.

In instances where parenteral administration is utilized, it may benecessary to employ somewhat bigger oligomers than those describedpreviously, with molecular weights ranging from about 500 to 30K Daltons(e.g., having molecular weights of about 500, 1000, 2000, 2500, 3000,5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).

The method may be used to treat any condition that may be remedied orprevented by administration of the particular conjugate. Those ofordinary skill in the art appreciate which conditions a specificconjugate may effectively treat. The actual dose to be administered mayvary depend upon the age, weight, and general condition of the subjectas well as the severity of the condition being treated, the judgment ofthe health care professional, and conjugate being administered.Therapeutically effective amounts are known to those skilled in the artand/or are described in the pertinent reference texts and literature.Generally, a therapeutically effective amount may range from about 0.001mg to 1000 mg, preferably in doses from 0.01 mg/day to 750 mg/day, andmore preferably in doses from 0.10 mg/day to 500 mg/day.

The unit dosage of any given conjugate may be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule may be known bythose of ordinary skill in the art or may be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

One advantage of administering the conjugates of the present inventionis that a reduction in first pass metabolism may be achieved relative tothe parent drug. Such a result is advantageous for many orallyadministered drugs that are substantially metabolized by passage throughthe gut. In this way, clearance of the conjugate can be modulated byselecting the oligomer molecular size, linkage, and position of covalentattachment providing the desired clearance properties. One of ordinaryskill in the art can determine the ideal molecular size of the oligomerbased upon the teachings herein. Preferred reductions in first passmetabolism for a conjugate as compared to the correspondingnonconjugated small drug molecule include: at least about 10%, at leastabout 20%, at least about 30; at least about 40; at least about 50%; atleast about 60%, at least about 70%, at least about 80% and at leastabout 90%.

Thus, the invention provides a method for reducing the metabolism of anactive agent. The method comprises the steps of: providing monodisperseor bimodal conjugates, each conjugate comprised of a moiety derived froma small molecule drug covalently attached by a stable linkage to awater-soluble oligomer, wherein said conjugate exhibits a reduced rateof metabolism as compared to the rate of metabolism of the smallmolecule drug not attached to the water-soluble oligomer; andadministering the conjugate to a patient. Typically, administration iscarried out via one type of administration selected from the groupconsisting of oral administration, transdermal administration, buccaladministration, transmucosal administration, vaginal administration,rectal administration, parenteral administration, and pulmonaryadministration.

All articles, books, patents, patent publications and other publicationsreferenced herein are incorporated by reference in their entireties.

EXPERIMENTAL

It is to be understood that while the invention has been described inconjunction with certain preferred and specific embodiments, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated. The preparation ofPEG-mers is described in, for example, U.S. Patent ApplicationPublication No. 2005/0136031.

¹H NMR (nuclear magnetic resonance) data was generated by a 300 MHz NMRspectrometer manufactured by Bruker.

Example 1 Synthesis of mPEG_(n)-5′-Floxuridine Monophosphate Conjugates

5′-O-TBDPS-floxuridine (1): Floxuridine (3.0 g, 12.2 mmol), DMAP (1.37g), and DIPEA (4.8 ml) were dissolved in anhydrous DMF (30 ml). TBDPSCl(3.15 ml) was added into the solution slowly at room temperature. Thereaction solution was allowed to stir overnight. Methanol (5 ml) wasadded. After 5 minutes, the solvent was evaporated under reducedpressure. The resulting residue was subjected to flash chromatography(MeOH/DCM=2%˜7%) to give the desired compound, 1 (4.9 g, 10.1 mmol,83%). ¹HNMR (DMSO-d₆): δ 7.89 (d, 1H), 7.65-7.62 (m, 4H), 7.50-7.42 (m,6H), 6.15 (t, 1H), 5.34 (d, 1H), 4.29-4.26 (m, 1H), 3.90-3.85 (m, 2H),3.77-3.73 (m, 1H), 2.20-2.16 (m, 2H), 1.01 (s, 9H).

3′-O—Ac-5′-O-TBDPS-floxuridine (2): 5′-O-TBDPS-floxuridine 1 (3.5 g, 7.2mmol) was dissolved in pyridine (8.1 ml, 100 mmol). Acetic anhydride(2.9 ml, 30 mmol)) was added slowly at room temperature. The reactionsolution was allowed to stir overnight. The reaction was worked up bywashing with sat. NaHCO₃ solution (50 ml). The mixed solution wasextracted with DCM (50 ml×3). The organic phases were combined and driedwith anhydrous Na₂SO₄. After filtering off the solid, the solvent wasremoved under reduced pressure to give compound 2 (3.5 g, 6.7 mmol,yield 92%), which was pure by TLC and used directly for next step.

3′-O—Ac-floxuridine (3): 3′-O—Ac-5′-O-TBDPS-floxuridine 2 (3.5 g, 6.7mmol) was dissolved in THF (30 ml). At room temperature, Bu₄NF (15 ml,1.0 M in THF, 15 mmol) was added into the solution. The reactionsolution was stirred at room temperature for 3 hrs. The solvent wasremoved under reduced pressure. The resulting residue was subjected toflash chromatography (CH₃OH/CH₂Cl₂=2%˜10%) to obtain compound 3 (1.8 g,6.2 mmol, yield 93%). ¹HNMR (DMSO-d₆): δ 11.86 (s, 1H), 8.21 (d, 1H),6.18-6.14 (m, 1H), 5.33-5.30 (m, 1H), 5.22-5.21 (m, 1H), 4.02-4.01 (m,1H), 3.66-3.63 (m, 2H), 2.29-2.27 (m, 2H), 2.06 (s, 3H).

3′-O—Ac-5′-(2-Cyanoethyl diisopropylphosphoramidite)-floxuridine (4):3′-O—Ac-floxuridine 3 (1.09 g, 4.0 mmol) and DIPEA (1.33 ml, 8 mmol)were dissolved in anhydrous DCM (50 ml). At room temperature,2-cyanoethyl diisopropylchlorophosphoramidite (1.15 ml, 5.0 mmol) wasadded to the solution dropwise. The solution was stirred for 30 minutesat room temperature and then washed with sat. NaHCO₃ solution (25 ml).The mixed solution was extracted with DCM (25 ml×3). The organic phaseswere combined and dried with anhydrous Na₂SO₄. After filtering offsolid, the solvent was removed under reduced pressure and the residue(2.4 g) was used directly for next step without further purification.

2-Cyanoethyl 5′-(2′-O—Ac-floxuridine) methoxy tri(ethylene glycol)phosphate (5a): Intermediate 4 (2.4 g) and tri(ethyleneglycol)monomethyl ether (787 mg, 4.8 mmol) were dissolved in anhydrousacetonitrile (50 ml). At room temperature, tetrazole solution (10.7 ml,0.45 M in acetonitrile, 4.8 mmol) was added into the reaction solution.The reaction solution was then stirred at room temperature for 4 hours.Iodine solution (110 ml, 0.1 M in THF/pyridine/H₂O 78:20:2) was added.After 10 minutes, Na₂S₂O₃ solution (5 g in 200 ml H₂O) was added. After5 minutes, the mixed solution was extracted with DCM (100 ml×3). Theorganic phase were combined and dried with anhydrous Na₂SO₄. Afterfiltering off solid, the solvent was removed under reduced pressure andthe resulting residue was subjected to flash chromatography(Acetone/EtOAc=10%˜70%) to obtain compound 5a (1.1 g, 1.9 mmol, yieldfor two steps, 48%). ¹HNMR (CDCl₃): δ 8.44 (s, 1H), 7.84 (d, 1H),6.35-6.31 (m, 1H), 5.35-5.32 (m, 1H), 4.44-4.34 (m, 7H), 3.74-3.39 (m,10H), 3.39 (s, 3H), 2.82 (t, 3H), 2.80-2.70 (m, 1H), 2.24-2.14 (m, 1H),2.13 (s, 3H).

2-Cyanoethyl 5′-(2′-O—Ac-floxuridine) methoxy hepta(ethylene glycol)phosphate (5b): Intermediate 4 (2.4 g) and hepta(ethyleneglycol)monomethyl ether (1.63 g, 4.8 mmol) were dissolved in anhydrousacetonitrile (50 ml). At room temperature, tetrazole solution (10.7 ml,0.45 M in acetonitrile, 4.8 mmol) was added into the reaction solution.The reaction solution was then stirred at room temperature for 4 hours.Iodine solution (110 ml, 0.1 M in THF/pyridine/H₂O 78:20:2) was added.After 10 minutes, Na₂S₂O₃ solution (5 g in 200 ml H₂O) was added. After5 minutes, the mixed solution was extracted with DCM (100 ml×3). Theorganic phases were combined and dried with anhydrous Na₂SO₄. Afterfiltering off solid, the solvent was removed under reduced pressure andthe resulting residue was subjected to flash chromatography(Acetone/EtOAc=30%˜90%) to obtain compound 5b (1.3 g, 1.7 mmol, yieldfor two steps 43%). ¹HNMR (CDCl₃): δ 7.78 (d, 1H), 6.32-6.27 (m, 1H),5.31-5.29 (m, 1H), 4.39-4.19 (m, 7H), 3.70-3.51 (m, 26H), 3.36 (s, 3H),2.82 (t, 3H), 2.80-2.70 (m, 1H), 2.22-2.19 (m, 1H), 2.09 (s, 3H).

5′-floxuridine methoxy tri(ethylene glycol) phosphate (6a): Compound 5a(1.1 g, 1.94 mmol) was dissolved in methanol (30 ml). At roomtemperature, sodium methoxide in methanol (25% wt, 2.0 ml) was addedinto the solution. The reaction solution was stirred for 4.5 hours atroom temperature. Dowex resin (hydrogen form, 50Wx4-200, 20 g) was addedand stirring continued for 10 minutes. The resin was filtered off andthe solvent was removed under reduced pressure. The resulting residuewas subjected to flash chromatography (MeOH/DCM=10%˜80%) to givecompound to give compound 6a (0.9 g, 1.90 mmol, yield 98%). ¹HNMR(MeOD): δ 7.94 (d, 1H), 6.30-6.26 (m, 1H), 4.46-4.44 (m, 1H), 4.26-4.16(m, 5H), 3.72-3.54 (m, 10H), 3.35 (s, 3H), 2.32-2.21 (m, 2H). LC/MS 473[M+H]⁺.

5′-floxuridine methoxy hepta(ethylene glycol) phosphate (6b): Compound5b (0.8 g, 1.1 mmol) was dissolved in methanol (20 ml). At roomtemperature, sodium methoxide in methanol (25% wt, 1.5 ml) was addedinto the solution. The reaction solution was stirred for 4.5 hours atroom temperature. Dowex resin (hydrogen form, 50Wx4-200, 18 g) was addedand stirring was continued for 10 minutes. The resin was filtered offand the solvent was removed under reduced pressure. The resultingresidue was subjected to flash chromatography (MeOH/DCM=10%˜80%) to givecompound 6b (0.65 g, 1.0 mmol, yield 91%). ¹HNMR (MeOD): δ 7.99 (d, 1H),6.29-6.27 (m, 1H), 4.47-4.46 (m, 1H), 4.22-4.09 (m, 5H), 3.71-3.59 (m,26H), 3.37 (s, 3H), 2.29-2.23 (m, 2H). LC/MS 649 [M+H]⁺.

Example 2 Synthesis of mPEG_(n)-5′-Gemcitabine Monophosphate Conjugates

N-Benzoyl-gemcitabine (1): A mixture of gemcitabine hydrochloride (300mg, 1 mmol), hexamethyldisilazane (5 ml), and a catalytic amount ofammonium sulfate (5 mg) in dioxane (5 ml) was heated under reflux for2.5 hours. The dioxane was evaporated and the reaction mixture wasdissolved in toluene (20 ml) and then evaporated three times. Afterremoval of toluene, the residue was dissolved in dichloromethane (10ml). To this solution was added N-methylimidazole (0.24 ml, 3 mmol) andbenzoyl chloride (0.35 ml, 3 mmol) and the solution was stirred forthree hours at room temperature. The reaction mixture was thenconcentrated to an oily residue, which was dissolved in a solution oftriethylamine (3 ml) and methanol (20 ml). This solution was stirred for1.5 hours at room temperature. The product was purified by flashchromatography (CH₂Cl₂/MeOH=2%˜10%) to give N-benzoyl-gemcitabine 1 (303mg, yield 83%). ¹H NMR (MeOD): δ 8.25 (d, 1H), 7.81-7.80 (m, 2H),7.56-7.22 (m, 4H), 6.14-6.04 (m, 1H), 4.21-4.10 (m, 1H), 3.83-3.80 (m,2H), 3.72-3.61 (m, 2H).

N-Benzoyl-5′-O-TBDPS-gemcitabine (2): N-Benzoyl-gemcitabine 1 (1.17 g,3.2 mmol), DMAP (1 mmol), and DIPEA (0.7 ml, 4.0 mmol) were dissolved inanhydrous DMF (30 ml). TBDPSCl (1.32 ml, 5.1 mmol) was added into thesolution slowly at room temperature and the solution was allowed to stirovernight. Methanol (5 ml) was added. After 5 minutes, the solvent wasremoved under reduced pressure. The resulting residue was subjected toflash chromatography (EtOAc/Haxanes=10%˜80%) to give compound 2 (1.5 g,2.5 mmol, yield 78%). ¹HNMR (CDCl₃): δ 8.10 (d, 1H), 7.91 (d, 2H),7.71-7.42 (m, 13H), 6.48-6.44 (m, 1H), 4.59-4.49 (m, 1H), 4.17-3.95 (m,4H), 1.13 (s, 9H).

N-Benzoyl-3′-O—Ac-5′-O-TBDPS-gemcitabine (3):N-Benzoyl-5′-O-TBDPS-gemcitabine 2 (1.5 g, 2.5 mmol) was dissolved inpyridine (8.1 ml, 100 mmol). Acetic anhydride (5.0 ml, 50 mmol)) wasadded slowly at room temperature and the solution was allowed to stirovernight. The reaction was worked up by washing with sat. NaHCO₃solution (50 ml). The mixed solution was extracted with DCM (50 ml×3).The organic phases were combined and dried with anhydrous Na₂SO₄. Afterfiltering off solid, the solvent was removed under reduced pressure togive compound 3 (1.5 g, 2.3 mmol, yield 92%), which was pure by TLC andthus used directly for next step.

N-Benzoyl-3′-O—Ac-gemcitabine (4):N-Benzoyl-3′-O—Ac-5′-O-TBDPS-gemcitabine 3 (1.5 g, 2.3 mmol) wasdissolved in THF (30 ml). At room temperature, a mixture of Bu₄NF (4.0ml, 1.0 M in THF, 4.0 mmol) and acetic acid (0.4 ml) was added into thesolution. The reaction solution was stirred at room temperature for 4hrs. The solvent was removed under reduced pressure. The residue wassubjected to flash chromatography (CH₃OH/CH₂Cl₂=2%˜10%) to obtaincompound 4 (0.82 g, 2.0 mmol, yield 87%). ¹HNMR (MeOD): δ 8.39-8.36 (d,1H), 8.02-7.99 (m, 2H), 7.70-7.54 (m, 4H), 6.42-6.37 (m, 1H), 5.52-5.45(m, 1H), 4.30-4.27 (m, 1H), 4.00-3.79 (m, 2H), 2.20 (s, 3H).

N-Benzoyl-3′-O—Ac-5′-(2-Cyanoethyldiisopropylphosphoramidite)-gemcitabine (5):N-benzoyl-3′-O—Ac-gemcitabine 4 (360 mg, 0.88 mmol) and DIPEA (0.25 ml,1.5 mmol) were dissolved in anhydrous DCM (50 ml). At room temperature,2-cyanoethyl diisopropylchlorophosphoramidite (0.28 ml, 1.2 mmol) wasadded to the solution dropwise. The solution was stirred at roomtemperature for 30 minutes and then washed with sat. NaHCO₃ solution (25ml). The mixed solution was extracted with DCM (25 ml×3). The organicphases were combined and dried with anhydrous Na₂SO₄. After filteringoff solid, the solvent was removed under reduced pressure and theresulting residue (530 mg) was used directly for next step withoutfurther purification.

2-Cyanoethyl 5′-(N-benzoyl-2′-O—Ac-gemcitabine) methoxy tri(ethyleneglycol) phosphate (6a): Intermediate 5 (530 mg) and tri(ethyleneglycol)monomethyl ether (164 mg, 1.0 mmol) were dissolved in anhydrousacetonitrile (10 ml). At room temperature, tetrazole solution (2.4 ml,0.45 M in acetonitrile, 1.1 mmol) was added into the reaction solution.The reaction solution was then stirred for 4 hours at room temperature.Iodine solution (20 ml, 0.1 M in THF/pyridine/H₂O 78:20:2) was added.After 10 minutes, Na₂S₂O₃ solution (2.5 g in 100 ml H₂O) was added.After 5 minutes, the mixed solution was extracted with DCM (100 ml×3).The organic phases were combined and dried with anhydrous Na₂SO₄. Afterfiltering off solid, the solvent was removed under reduced pressure andthe resulting residue was subjected to flash chromatography(acetone/EtOAc=10%˜50%) to obtain compound 6a (280 mg, 0.41 mmol, yieldfor two steps, 47%). ¹HNMR (CDCl₃): δ 7.95-7.87 (m, 3H), 7.58-7.53 (m,2H), 7.48-7.43 (m, 2H), 6.45-6.40 (m, 1H), 5.40-5.35 (m, 1H), 4.42-4.26(m, 7H), 3.63-3.50 (m, 10H), 3.30 (s, 3H), 2.80-2.76 (m, 2H), 2.15 (s,1H).

2-Cyanoethyl 5′-(N-benzoyl-2′-O—Ac-gemcitabine) methoxy hepta(ethyleneglycol) phosphate (6b): Intermediate 5 (677 mg) and hepta(ethyleneglycol)monomethyl ether (463 mg, 1.3 mmol) were dissolved in anhydrousacetonitrile (15 ml). At room temperature, tetrazole solution (3.1 ml,0.45 M in acetonitrile, 1.4 mmol) was added into the reaction solution.The reaction solution was then stirred for 4 hours at room temperature.Iodine solution (26 ml, 0.1 M in THF/pyridine/H₂O 78:20:2) was added.After 10 minutes, Na₂S₂O₃ solution (2.5 g in 100 ml H₂O) was added.After 5 minutes, the mixed solution was extracted with DCM (100 ml×3).The organic phases were combined and dried with anhydrous Na₂SO₄. Afterfiltering off solid, the solvent was removed under reduced pressure andthe resulting residue was subjected to flash chromatography(acetone/EtOAc=10%=80%) to obtain compound 6b (610 mg, 0.7 mmol, yieldfor two steps 55%). ¹HNMR (CDCl₃): δ 7.98-7.89 (m, 3H), 7.60-7.57 (m,2H), 7.49-7.45 (m, 2H), 6.45-6.40 (m, 1H), 5.42-5.36 (m, 1H), 4.44-4.28(m, 7H), 3.65-3.51 (m, 26H), 3.32 (s, 3H), 2.81-2.77 (m, 2H), 2.17 (s,1H).

5′-gemcitabine methoxy tri(ethylene glycol) phosphate hydrochloride(7a): Compound 6a (280 mg, 0.41 mmol) was dissolved in methanol (10 ml).At room temperature, ammonia in methanol (4.5 ml, 7 N, 31.5 mmol) wasadded into the solution. The reaction solution was stirred at roomtemperature for 18 hours. The solvent was evaporated under reducedpressure. The resulting residue was dissolved in methanol (5 ml). HCl inethyl ether (1 N, 1 ml) was then added to the solution. The solvent wasremoved under reduced pressure and the crude residue was subjected toflash chromatography (MeOH/DCM=10%˜80%) to give compound 7a (110 mg,0.22 mmol, yield 54%). ¹HNMR (MeOD): δ 8.02 (d, 1H), 6.26 (t, 1H), 6.16(d, 1H), 4.35-4.01 (m, 6H), 3.71-3.54 (m, 10H), 3.38 (s, 3H). LC/MS: 490[M+H]⁺.

5′-gemcitabine methoxy hepta(ethylene glycol) phosphate hydrochloride(7b): Compound 6b (600 mg, 0.70 mmol) was dissolved in methanol (40 ml).At room temperature, ammonia in methanol (18 ml, 7 N, 126 mmol) wasadded into the solution. The reaction solution was stirred at roomtemperature for 18 hours. The solvent was evaporated under reducedpressure. The resulting residue was dissolved in methanol (5 ml). HCl indioxane (4 N, 0.7 ml) was then added to the solution. The solvent wasremoved under reduced pressure and the crude residue was subjected toflash chromatography (MeOH/DCM=10%˜80%) to give compound 7b (320 mg,0.48 mmol, yield 69%). ¹HNMR (MeOD): δ 8.07 (d, 1H), 6.33 (d, 1H), 6.22(t, 1H), 4.44-4.19 (m, 6H), 3.69-3.56 (m, 26H), 3.38 (s, 3H). LC/MS: 666[M+H]⁺.

Example 3 Synthesis of Di-PEG-Floxuridine Phosphate Conjugates

5′-O-TBDPS-floxuridine (1)

Floxuridine (3.0 g, 12.2 mmol), DMAP (1.37 g), and DIPEA (4.8 ml) weredissolved in anhydrous DMF (30 ml). TBDPSCl (3.15 ml) was added into thesolution slowly at room temperature (RT). The reaction solution wasstirred at RT overnight. Methanol (5 ml) was added. After 5 minutes, thesolvent was evaporated at reduced pressure. The residue was subjected toflash chromatography (MeOH/DCM=2%˜7%) to obtain compound 1 (4.9 g, 10.1mmol, 83%). ¹H NMR (DMSO-d₆): δ 7.89 (d, 1H), 7.65-7.62 (m, 4H),7.50-7.42 (m, 6H), 6.15 (t, 1H), 5.34 (d, 1H), 4.29-4.26 (m, 1H),3.90-3.85 (m, 2H), 3.77-3.73 (m, 1H), 2.20-2.16 (m, 2H), 1.01 (s, 9H).

3′-O—Ac—S′—O-TBDPS-floxuridine (2)

5′-O-TBDPS-floxuridine 1 (3.5 g, 7.2 mmol) was dissolved in pyridine(8.1 ml, 100 mmol). Acetic anhydride (2.9 ml, 30 mmol)) was added slowlyat RT. The reaction solution was stirred at RT overnight. The reactionwas worked up by washing with sat. NaHCO₃ solution (50 ml). The mixedsolution was extracted with DCM (50 ml×3). The organic phases werecombined and dried with anhydrous Na₂SO₄. After filtering off the solid,the solvent was evaporated at reduced pressure to give compound 2 (3.5g, 6.7 mmol, yield 92%), which was pure on TLC and used directly withoutfurther purification in the next step.

3′-O—Ac-floxuridine (3)

3′-O—Ac-5′-O-TBDPS-floxuridine 2 (3.5 g, 6.7 mmol) was dissolved in THF(30 ml). At RT, Bu₄NF (15 ml, 1.0 M in THF, 15 mmol) was added into thesolution. The reaction solution was stirred at RT for 3 hrs. The solventwas evaporated at reduced pressure. The resulting residue was subjectedto flash chromatography (CH₃OH/CH₂Cl₂=2%˜10%) to obtain compound 3 (1.8g, 6.2 mmol, yield 93%). ¹HNMR (DMSO-d₆): δ 11.86 (s, 1H), 8.21 (d, 1H),6.18-6.14 (m, 1H), 5.33-5.30 (m, 1H), 5.22-5.21 (m, 1H), 4.02-4.01 (m,1H), 3.66-3.63 (m, 2H), 2.29-2.27 (m, 2H), 2.06 (s, 3H).

3′-O—Ac-5′-[di-methoxy tri(ethylene glycol)phosphate]-floxuridine (4a)

3′-O—Ac-floxuridine 3 (170 mg, 0.62 mmol) was dissolved in anhydrouspyridine (3 ml). At −40° C. under N₂, POCl₃ (0.08 ml, 0.88 mmol) wasadded to the solution. The solution was stirred at −40° C. for 15minutes. Tri(ethylene glycol)monomethyl ether (394 mg, 2.4 mmol) wasthen added to solution. The reaction solution was stirred and allowed towarm to room temperature slowly over 4 hours. The solvent was evaporatedat reduced pressure. The residue was used for next step directly withoutfurther purification.

3′-O—Ac-5′-[di-methoxy hepta(ethylene glycol)phosphate]-floxuridine (4b)

3′-O—Ac-floxuridine 3 (190 mg, 0.70 mmol) was dissolved in anhydrouspyridine (3 ml). At −40° C. under N₂, POCl₃ (0.07 ml, 0.77 mmol) wasadded to the solution. The solution was stirred at −40° C. for 15minutes. Hepta(ethylene glycol)monomethyl ether (850 mg, 2.5 mmol) wasthen added to solution. The reaction solution was stirred and allowed towarm to room temperature slowly over 4 hours. The solvent was evaporatedat reduced pressure. The residue was used for next step directly withoutfurther purification.

5′-floxuridine di-methoxy tri(ethylene glycol) phosphate (5a)

Compound 4a was dissolved in methanol (10 ml). At RT, sodium methoxidein methanol (0.1 N, 14 ml) was added to the solution. The reactionsolution was stirred at RT for 1 hour. Then 1.0 N HCl (1.6 ml) wasadded. The solvent was evaporated. The residue was subjected to flashchromatography (MeOH/DCM=2%˜10%) to give compound 5a (300 mg, 0.49 mmol,yield 69%). ¹HNMR (CDCl₃): δ 8.47 (m, 1H), 7.82 (d, 1H), 6.28 (t, 1H),4.64-4.62 (m, 1H), 4.43-4.22 (m, 6H), 4.09-4.05 (m, 1H), 4.00-3.97 (m,1H), 3.74-3.54 (m, 20H), 3.40 (s, 6H), 2.43-2.41 (m, 1H), 2.21-2.16 (m,1H). LC/MS 619 [M+H]⁺.

5′-floxuridine di-methoxy hepta(ethylene glycol) phosphate (5b)

Compound 4b was dissolved in methanol (10 ml). At RT, sodium methoxidein methanol (0.5 N, 4 ml) was added to the solution. The reactionsolution was stirred at RT for 2 hours. Then 1.0 N HCl (2.2 ml) wasadded. The solvent was evaporated. The residue was subjected to flashchromatography (MeOH/DCM=3%˜10%) to give compound 5a (375 mg, 0.39 mmol,yield 55%). ¹HNMR (CDCl₃): δ 8.84 (m, 1H), 7.82 (d, 1H), 6.28 (t, 1H),4.60-4.57 (m, 1H), 4.40-4.22 (m, 6H), 4.09-4.05 (m, 1H), 3.97-3.94 (m,1H), 3.74-3.54 (m, 52H), 3.39 (s, 6H), 2.41-2.39 (m, 1H), 2.21-2.15 (m,1H). LC/MS 971 [M+H]⁺.

These and other compounds of the invention may be similarly synthesizedusing the methods described herein, as well as using techniques known toone skilled in the art.

Example 4

The NS5B protein of hepatitis C virus (HCV) contains the RNA-dependentRNA polymerase that is the catalytic component of the HCV replicationmachinery. Since NS5B polymerase synthesizes RNA from an RNA template,selective molecules, shown below, have been generated to inhibit theproduction of viral genomic DNA. Attaching a water-soluble, non-peptidicpolymer can improve these inhibitors further. The benefits of PEGylationhere are: improved oral bioavailability (˜4%), decreased dose (e.g. 3000mg daily doses), reduced metabolism, and delivery of the activemonophosphate form which then gets phosphorylated to the di- andtri-phosphates.

Synthesis of Pathway for PEG-NS5B Polymerase Inhibitor, PSI-6130

Similarly, a water-soluble, non-peptidic polymer can be attached tocompounds mentioned above, and other compounds, e.g.:

or R1626 which is a pro-drug form of R1479.

PSI-6130, as shown,

or R7128 which is a pro-drug form of PSI-6130.

Valopicitabine, shown below, is a nucleoside analogue and the orallybioavailable prodrug of NM107 that competitively inhibits the NS5Bpolymerase, causing chain termination.

Example 5 In Vitro Screening of PEG-Floxuridine Monophosphate inSelected Human Cancer Cell Lines Experimental Design

Test articles: Pegylated FloxuridinesTest system: Human cancer cell lines—Colon (HT29), Ovary (A2780), Lung(HOP62), and Prostate (DU145) Breast (MCF-7).Reference drug: FloxuridineDose levels: Four concentrations (10-4-10-7 Molar concentration) intriplicate.Method: Sulforhodamine B (SRB) assay with 48 h drug exposure

Reference: Nature Protocols 1, 1112-1116 (2006) Conjugates

mono-mPEG₃-5′-Gemcitabine monophosphatemono-mPEG₇-5′-Gemcitabine monophosphatedi-mPEG₃-5′-Floxuridine monophosphatedi-mPEG₇-5′-Floxuridine monophosphatemono-mPEG₃-5′-Floxuridine monophosphatemono-mPEG₇-5′-Floxuridine monophosphateIn vitro screening using Colon (HT29), Ovary (A2780), Lung (HOP62), andProstate(DU 145) tumor cell lines revealed that the tested conjugates possessgrowth inhibiting(GI₅₀) activity relative to Floxuridine.The GI₅₀ values of mono-mPEG₇-Floxuridine monophosphate were 3.1×10- and2.2×10⁻⁶ in Hop62 and MCF7 respectively.The conjugates showed no conversion (hydrolysis) to Floxuridine in thecell media during experimentation.

In Vitro Screening of PEG-Gemcitabine Monophosphate in Selected HumanCancer Cell Lines

TABLE 1 GI₅₀ (molar concentration) in Cell Line NCI/ADR- Test ArticleDU145 MCF7 HT29 COLO205 HCT15 A2780 Hop62 RES Gemcitabine <1 × 10⁻⁷ 1.6× 10⁻⁷ 3.2 × 10⁻⁵ 1.8 × 10⁻⁷ 2.3 × 10⁻⁶ 1.5 × 10⁻⁷ 2.4 × 10⁻⁶ 2.0 × 10⁻⁶Mono (mPEG₇) Gemcitabine <1 × 10⁻⁷ 1.6 × 10⁻⁷ 2.8 × 10⁻⁵ 1.6 × 10⁻⁷ 2.2× 10⁻⁶ 1.5 × 10⁻⁷ 2.6 × 10⁻⁶ 1.7 × 10⁻⁷ Mono (mPEG₃) Gemcitabine Not  <1× 10⁻⁷ 2.1 × 10⁻⁶ 1.4 × 10⁻⁷ 1.6 × 10⁻⁷  <1 × 10⁻⁷ 1.8 × 10⁻⁶ Notattained attainedSimilarly, activities of the compounds of the present invention mayfurther be tested in following cell lines: HCT-116, HT-29 and CAKI-1,DLD-1, HCT-116, HT-29, SW-620, NCI-H23, NCI-H460, NCI-H522, PANC-1,HL-60, CCRF-CEM, K-562, As283, and RL cells.

Stability in Cell Culture Medium

The MCF-7 cell line was grown in RPMI 1640 medium containing 5% fetalbovine serum and 2 mM L-glutamine. Cells were inoculated into 24-wellmicrotiter plates in 100 μL at 5×10³ cells/well, in duplicate. After 24h, test articles were added to the cells for 48 h, after which time thesupernatant from each of the wells was collected and stored below −15°C. until HPLC analysis. The frozen samples were thawed and homogenizedby vortex mixing. A 10 μl aliquot of the sample was transferred into aclean EPPENDORF tube. Samples were diluted with 190 μl methanol: watermixture (1:1, v/v). The tubes were vortex mixed and centrifuged for 10min. The supernatant was transferred to HPLC vials and 10 μl volume ofthe sample was injected onto the HPLC system. A calibration curve wasgenerated by spiking known amounts of floxuridine in cell culture media,because the retention time of floxuridine was decreased by the presenceof medium (7.25 min vs. 9.2 min).

TABLE 2 Summary of HPLC data: peak area of measured floxuridine DilutionTotal factor Calculated (10 μL Concentra- Calculated sample tion in theNominal Concen- diluted sample using concen- Peak tration to dilutiontration Test Article Area (μg/mL) 200 μL) factor (μg/mL) Floxuridine119819 1.211 20 24.22 24.6* Floxuridine — NA 20 NA NA Mono (mPEG3)Floxuridine — NA 20 NA NA Mono (mPEG3) Floxuridine — NA 20 NA NA Di(mPEG3)The concentration of floxuridine estimated in culture medium after cellswere treated with floxuridine, and three floxuridine derivatives were24.44, and below the assay detection level, respectively. Thus,floxuridine is not released from PEG-floxuridine in cell culture medium.

In Vitro Activity Studies

The aim was to determine the GI₅₀ values for PEG-floxuridine conjugatesagainst a panel of cell lines, and based on previous data from Xenograftstudies. The cells were exposed for 24 h, and cell viability assessed byusing the Sulphorhodamine (SRB) assay. The test articles weresolubilized in dimethylsulfoxide and were tested at four different molar(M) concentrations; 10⁻⁴, 10⁻⁵, 10⁻⁶, M. GI₅₀ (the concentration neededto reduce the growth of treated cells to half that of untreated controlcells) was calculated for each condition([(T_(i)−T_(z))/(C-T_(z))]×100=50, where T_(i), T_(z), and C refer toabsorbance values obtained from each tested concentration, time zero,and control treatment, respectively).

TABLE 3 GI₅₀ values (μM) of floxuridine and PEG conjugates in HumanTumor Cell lines of Prostate, Colon, Ovary, Lung and Breast. NCI/ADR-Compound DU145 MCF7 HT29 COLO205 HCT15 A2780 Hop62 RES Floxuridine 260.15 25 ND ND 0.065 0.24 ND Floxuridine 0.12 0.11 >100 1.8 32 2.1 >100<0.1 Mono (mPEG3) Floxuridine 0.13 0.13 ~>100 1.9 ~32 2.3 >100 <0.1 Mono(mPEG7) Floxuridine >100 >100 >100 ND ND >100 >100 ND Di (mPEG3)Floxuridine >100 >100 >100 ND ND >100 >100 ND Di (mPEG7) ratio mono0.005 0.7 4 ND ND 32 417 ND (mPEG3) vs flox ratio mono 0.005 0.9 4 ND ND35 417 ND (mPEG7) vs flox ratio 4 667 4 ND ND 1538 417 ND di(mPEG3) vsflox ratio 4 667 4 ND ND 1538 417 ND di(mPEG7) vs flox

The mono-PEG-floxuridine monophosphate derivatives and/or floxuridine(no data for 3 cell lines) inhibited the growth of 5 of 8 cell lineswith GI₅₀ values from the nM to low μM range (DU145 prostate, MCF-7 andNCI/Adr-Res breast, COL0205 colon, and A2780 ovarian). However, some ofthe GI₅₀ values should be interpreted with caution because theconcentration range tested was not centered around the GI₅₀. The di-PEGderivatives were inactive in all cell lines tested. In conclusion, thereis a range of sensitivities, and fold response to PEGylated floxuridinesversus floxuridine.

In Vitro Cytotoxicity Assays

The objective of these studies were to determine the IC₅₀ values forPEG-floxuridine conjugates against floxuridine-resistant cell linesbased on data from previous Xenograft studies. The cells were exposedfor 72 h, and cell viability assessed by using the CellTiter-Glo assay.

TABLE 4 IC₅₀- values of floxuridine, NKT-10154, and NKT-10156 obtainedfrom curve fits of data following exposure of resistant cell lines IC₅₀(nM) Ratio vs floxuridine Flox- Flox- Flox- Flox- uridine uridineuridine uridine Flox- Mono Mono Mono Mono Cell Line uridine (mPEG3)(mPEG7) (mPEG3) (mPEG7) HCT-116 32 56 48 2 2 HT-29 90 87 76 1 1 CAKI-1110 145 157 1 1 MIA PaCa-2 54 64 96 1 2The PEGylated floxuridine monophosphate derivatives and floxuridineinhibited the growth of all of the tested cell lines with IC₅₀ values inthe nM range. Like the previous study, some of the IC₅₀ values should beinterpreted carefully because the concentration range tested was notcentered around the IC₅₀.

Bi-Directional Permeability of PEG-Floxuridine Conjugates in Caco-2 andMDCK Cells

TABLE 5 Recovery and Apparent Permeability (10⁻⁶ cm/s) of test compoundspercent recovery P_(app) Papp B-A/ Absorption Significant A -> B B -> AA -> B B -> A Papp A-B potential efflux floxuridine Caco-2 75 91 0.751.47 2.0 Low No MDCK 50 82 <2.46 <2.70 ND — No NKT-10154 Caco-2 96 94<0.19 <0.25 ND Low No MDCK 98 98 <0.06 <0.07 ND — No NKT-10156 Caco-2 7989 0.14 0.06 0.4 Low No MDCK 100 90 <0.06 <0.08 ND — No CapecitabineCaco-2 110 99 2.22 8.71 3.9 High Yes MDCK 104 99 0.16 0.14 0.9 — NoThe absorption potential of the compounds was determined in Caco-2 andMDCK cells, where absorption is considered low if P_(app) (A-B) is<1.0×10⁻⁶ cm/s. An orally bioavailable nucleoside analog, Capecitabine,showed high permeability across Caco-2 monolayers, but relatively poorpermeability through MDCK monolayers. Floxuridine and PEGylatedfloxuridine compounds showed poor permeability through both cell lines.Floxuridine had poor analytical sensitivity compared to the otheranalytes (LOD was approximately 0.03 uM, compared to <0.01 uM,respectively).

Gemcitabine: In Vitro Stability Studies Stability in Cell Culture Medium(LS-2007-016)

The MCF-7 cell line was grown in RPMI 1640 medium containing 5% fetalbovine serum and 2 mM L-glutamine. This cell lines was chosen because itwas moderately sensitive to gemcitabine (GI₅₀˜100 nM). Cells wereinoculated into 24-well microtiter plates in 100 μL at 5×10³ cells/well,in duplicate. After 24 h, test articles were added to the cells for 24or 48 h, after which time the supernatant from each of the wells wascollected and stored below −15° C. until LC/MS analysis.

Gemcitabine showed roughly 100-110% recovery in all media types. ForPEG-gemcitabine, there are two “bins” of data: 60-80% recovery, versus100% recovery of PEG-gemcitabine. Samples from RPMI+FBS, RPMI+FBS+cells,and conditioned medium, where 10% FBS was a component, had 60-80%recovery, whereas RPMI alone or with “serum replacement” showed 100-120%recovery. Samples from PEG-gemcitabine incubated in PBS hadapproximately 80% recovery of test article at both time points. It isnot clear why the stability of PEG-gem is lower in PBS versus RPMImedium. On a mM basis, about 80% of the mPEG3 or mPEG7 remains after 24and 48 h, thus there is not a significant decrease over time. The <10%decrease in PEG-gemcitabine that is observable from time point 24 to 48h is due to conversion to gemcitabine.

In Vitro Activity Studies

The objective was to determine the IC₅₀ values for PEG-gemcitabineconjugates against a panel of cell lines, and based on previous datafrom Xenograft studies. The cells were exposed for 24 h, and cellviability assessed by using the Sulphorhodamine (SRB) assay. The testarticles were solubilized in dimethyl sulfoxide and were tested at fourdifferent molar (M) concentrations; 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷ M. GI₅₀ (theconcentration needed to reduce the growth of treated cells to half thatof untreated control cells) was calculated for each condition([(T_(i)−T_(z))/(C−T_(z))]×100=50, where T_(i), T_(z), and C refer toabsorbance values obtained from each tested concentration, time zero,and control treatment, respectively).

TABLE 7 GI₅₀ values (μM) of gemcitabine and its PEG conjugates in HumanTumor Cell lines of Colon, Ovary, Lung and Breast. NCI/ COLO- ADR- DU145MCF7 HT29 205 HCT15 A2780 Hop62 RES Gemcitabine <0.1 0.16 32 0.18 2.30.15 2.4 2.0 Mono (mPEG7) Gemcitabine <0.1 0.158 28 0.16 2.2 0.15 2.61.7 Mono (mPEG3) Gemcitabine <<0.1 <0.1 2.1 0.14 0.16 <0.1 1.8 nd rationd 2 15 1 14 2 1 nd mPEG7 vs gem ratio nd 2 13 1 14 2 1 nd mPEG3 vs gem

The PEG-gemcitabine derivatives and gemcitabine inhibited the growth ofmost of the cell lines with GI₅₀ values from the nM to low μM range,except for HT-29. Some of the GI₅₀ values should be interpreted withcaution because the concentration range tested was not centered aroundthe GI₅₀. Adriamycin was used as a positive control.

In Vitro Cytotoxicity Assays

The objective of these studies was to determine the IC₅₀ values forPEG-gemcitabine conjugates against gemcitabine-resistant cell linesbased on data from previous Xenograft studies. The cells were exposedfor 72 h, and cell viability assessed by using the CellTiter-Glo assay.

TABLE 8 IC₅₀- values of gemcitabine, NKT-10238, and NKT-10239 obtainedfrom curve fits of data following exposure of resistant cell lines. IC₅₀(nM) Ratio vs gem mPEG3- mPEG7- mPEG3- mPEG7- Cell Line Gemcitabine GemGem Gem Gem HCT-116 1.9 2.9 4 2 2 HT-29 111 755 6252 7 56 CAKI-1 8.3 2150 3 6 MIA 26 76 84 3 3 PaCa-2 DLD-1 22 93 36 4 2 SW-620 49 95 182 2 4NCI-H23 6.9 18 47 3 7 NCI- 14 43 48 3 3 H460 NCI- 4.4 36 26 8 6 H522PANC-1 >1,000,000 >1,000,000 >1,000,000 na na HL-60 1.7 2 2.6 1 2 CCRF-7 6.5 10 1 1 CEM K-562 697 914 1148 1 2 AS283 124 408 398 3 3 RL 281 568259 2 1

The PEG-gemcitabine derivatives and gemcitabine inhibited the growth ofmost of the cell lines with IC₅₀ values from the nM to low μM range,except for PANC-1 cells. Like the previous study, some of the IC₅₀values should be interpreted carefully because the concentration rangetested was not centered around the IC₅₀. Resistance was observed in onecell line, PANC-1 (mechanism unknown), and there was no significantdifference in cell growth after treatment with gemcitabine or thePEGylated derivatives. The PANC-1 pancreatic cell line, in contrast toBxPC-3, Capan-1, or MIA-PaCa-2, has been shown previously to beresistant in vitro to gemcitabine, and 4′-thio-FAC, a deoxycytidinesulfur- and fluoro-substituted analog (Zajchowski et al., (2005). Int JCancer. 114(6), 1002-9).

Bi-Directional Permeability of PEG-Gemcitabine Conjugates in Caco-2 andMDCK Cells

The absorption potential of the compounds was determined in Caco-2 andMDCK cells, where absorption is considered low if P_(app) (A-B) is<1.0×10⁻⁶ cm/s. An orally bioavailable nucleoside analog, Capecitabine,showed high permeability across Caco-2 monolayers, but relatively poorpermeability through MDCK monolayers. Gemcitabine and PEGylatedgemcitabine compounds showed poor permeability through both cell lines.

TABLE 9 Recovery and Apparent Permeability (10⁻⁶ cm/s) of test compoundspercent recovery P_(app) Papp B-A/ Absorption Significant A -> B B -> AA -> B B -> A Papp A-B potential efflux gemcitabine Caco-2 90 89 0.30.42 1.4 Low No MDCK 96 95 0.17 0.12 0.7 — No mPEG3-Gem Caco-2 78 90<0.06 <0.08 ND Low No MDCK 91 96 <0.06 <0.08 ND — No mPEG7-Gem Caco-2 7486 <0.06 <0.08 ND Low No MDCK 94 96 <0.06 <0.08 ND — No CapecitabineCaco-2 110 99 2.22 8.71 3.9 High Yes MDCK 104 99 0.16 0.14 0.9 — No

1. A compound having the following structurebase-sugar-phosphate-[X]_(b)-[POLY]_(a) wherein: “base-sugar-” has thefollowing structure

X is a linker when present; (b) is an integer having a value of zero orone; POLY is a water-soluble, non-peptidic oligomer having anend-capping group selected from hydroxyl and carbon-containingend-capping groups; and (a) is an integer having a value of one or two,inclusive. 2-17. (canceled)
 18. The compound of claim 1, wherein thewater-soluble, non-peptidic oligomer is a poly(alkylene oxide).
 19. Thecompound of claim 18, wherein the poly(alkylene oxide) is apoly(ethylene oxide).
 20. The compound of claim 1, whereinwater-soluble, non-peptidic oligomer is made of between 2 and 30monomers.
 21. The compound of claim 20, wherein the water-soluble,non-peptidic oligomer is made of between 2 and 10 monomers.
 22. Thecompound of claim 18, wherein the poly(alkylene oxide) includes analkoxy or hydroxy end-capping moiety.
 23. The compound of claim 1,wherein a single water-soluble, non-peptidic oligomer is attached to thenucleoside phosphate residue.
 24. The compound of claim 1, wherein morethan one water-soluble, non-peptidic oligomer is attached to thenucleoside phosphate residue.
 25. The compound of claim 1, wherein thenucleoside phosphate residue is covalently attached via a stablelinkage.
 26. The compound of claim 1, wherein the nucleoside phosphateresidue is covalently attached via a degradable linkage.
 27. Thecompound of claim 1, wherein the linkage is an ether linkage.
 28. Thecompound of claim 1, wherein the linkage is an ester or a phosphoesterlinkage.
 29. A composition comprising a compound claim 1, andoptionally, a pharmaceutically acceptable excipient.
 30. A compositionof matter comprising a compound of claim 1, wherein the compound ispresent in a dosage form.
 31. (canceled)
 32. (canceled)
 33. The compoundof claim 1, having the following structure:

wherein (n) is an integer between 2 and 30 and Me is methyl.
 34. Thecompound of claim 33, wherein (n) is
 3. 35. The compound of claim 33,wherein (n) is 7.